Method for enhancing transport of gases to tissues

ABSTRACT

A method is disclosed for the treatment of right to left shunt. The method comprises introducing, into the blood circulation of an individual, to be treated, a therapeutically effective amount of the stabilized microbubbles. In a preferred embodiment, oxygen is additionally administered to the individual.

This application is a continuation-in-part of our U.S. application Ser.No. 08/753,581 filed Nov. 26, 1996, now U.S. Pat. No. 5,869,538 thedisclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to methods of using various gascarriers to enhance the transport of gases, such as oxygen, inert gases,anesthetic gases, or toxic gases, to or from body tissues. Morespecifically, the invention relates to methods of using gas carriers,wherein the gas carriers are in a form comprising stabilizedmicrobubbles, to enhance delivery or removal of one or more gases to orfrom body tissues when such delivery or removal is desirable for medicalreasons. The methods are especially useful for enhancing the transportof oxygen by one or more species of oxygen carriers for applications orconditions such as hemorrhagic anemia, inactivated hemoglobin, organperfusion, coronary angioplasty, enhancement of oxygen concentration inarterial blood under conditions of right to left shunting from thevenous to arterial side of the systemic circulation, oxygenation ofischemic tissues resulting from vascular obstructions, and oxygenationof tissues for cancer radiation and chemotherapy. The methods are alsouseful for enhancing transport of gases by one or another species of gascarriers for applications such as prevention or treatment ofdecompression sickness and gas embolism, and removal of toxic gases fromsolution in body tissues.

BACKGROUND OF THE INVENTION

Because blood is prone to viral contamination, and because donated bloodhas a limited shelf life, donated blood appears to be in constant shortsupply. In response, much effort has been focused on the development ofcompositions commonly referred to as "blood substitutes" or "artificialblood". Such compositions transport oxygen to tissues, as do red bloodcells, but such compositions generally lack the ability to perform themetabolic, regulatory, and protective functions of blood. Thus, thesecompositions are more appropriately termed "gas carriers".

Further, in certain clinical situations, it is desirable to supplementthe amount of oxygen carried by an individuals blood. One situationwhere oxygen supplementation is desirable is during the phenomenon ofshunting. Clinically significant right-to-left shunting of blood betweenthe right and left atria or ventricles of the heart occurs, for example,in pneumonia and in conditions involving septal defects. In thesesituations, which are characterized by arterial hypoxia, hypercapnia andacidosis, some of the deoxygenated venous blood is not subject toreoxygenation in the lungs before returning to the left heart and thesystemic circulation. For example, in severe pneumonia, alveoli canbecome filled with cells and other material due to the inflammatoryprocess resulting in the lack of air flow and hence lack of oxygendelivery to these alveoli. As a consequence, the oxygen poor venousblood supplying the alveoli is not oxygenated adequately. Theoxygen-poor blood mixes with the adequately oxygenated blood fromventilated alveoli. The net result of this mixing of shunted oxygen-poorblood and oxygenated blood is that the mixed blood going to the systemicarteries is less oxygenated than normal leading to inadequate oxygensupply to body tissues. Various gas carriers are known to those skilledin the art. One class of gas carriers includes hemoglobin, modifiedhemoglobin (polymerized, conjugated, crosslinked, orphospholipid-encapsulated), recombinant hemoglobin, and hemoglobinderivatives. Another class of gas carriers comprises liquidperfluorochemicals. Typically, perfluorochemicals (PFCS; also known as"perfluorocarbons") are liquids that dissolve oxygen and that arecomposed of 8 to 10 carbon atoms per molecule. Oxygen, carbon dioxide,and nitrogen are examples of gases that are highly soluble in PFCs. Forintravascular use, PFCs are generally administered in emulsions, withemulsifiers such as egg yolk phospholipid or poloxamers, because PFCsare insoluble in water or an aqueous environment. However, depending onthe factors such as particle size, viscosity, surface tension (in caseof bubbles), and chemical composition of the PFC emulsion, stability inthe presence of blood and other biological fluids andefficiency/efficacy of gas transport to tissues varies. As a result,various liquid PFC emulsions are being investigated for characteristicssuch as elimination, distribution, tissue retention, and physiologicalchanges after administration. Such factors and characteristics affectthe ability of PFCs to be used in various medical applications. Studiesto date indicate that significant amounts of PFC emulsion need beinjected to sufficiently supplement or replace the oxygen carryingcapacity of hemoglobin. Further, in the body, many liquid PFC emulsionsbecome sequestered in organs such as the spleen and liver.

Emulsions of liquid PFCs have been described to be potentially useful asoxygen carriers for various medical applications including as a "bloodsubstitute" in the treatment of heart attack, stroke, and organperfusion; as adjuvants to coronary angioplasty; and in cancer radiationtreatment and chemotherapy. PFCs said to be useful in such applicationsare described, for example, in U.S. Pat. Nos. 5,403,575; 4,868,318;4,866,096; 4,865,836; 4,686,024; 4,534,978; 4,443,480; 4,423,077;4,252,827; 4,187,252; 4,186,253; 4,110,474; and 3,962,439. Such liquidPFC emulsions include perfluorooctyl bromide, perfluorooctyl dibromide,bromofluorocarbons, perfluoroethers, Fluosol DA™, F-44E,1,2-bisperfluorobutyl-ethylene, F-4-methyl octahydroquinolidizine, 9 to12 carbon perfluoro amines, perfluorodecalin, perfluoroindane,perfluorotrimethyl adamantane.

Recently, microbubbles have been developed for use as contrast-enhancingagents for ultrasonic imaging of the heart and blood vessels. Onecommercially available preparation of such microbubbles is produced bysonication of albumin solution (see for example U.S. Pat. Nos.4,718,433; 4,774,958 and 4,957,656). These microspheres are made bysonicating protein solutions, such as 5% human albumin. A method ofpreparing stable suspensions of microbubbles using various proteinsolutions, and their use in echographic investigation is also disclosedin U.S. Pat. No. 5,310,540. Another species, lipid-coated microbubbles,their method of preparation, and their use in ultrasound and magneticresonance imaging techniques, is described in U.S. Pat. No. 5,215,680. Amethod of making an ultrasound contrast agent, comprising gas-filledmicrobubbles wherein the gas is a halogenated hydrocarbon, with improvedresistance against collapse due to pressure is disclosed in U.S. Pat.No. 5,413,774.

Other microbubbles are formed from PFCs (U.S. Pat. No. 5,409,688) inmethods for ultrasound imaging (U.S. Pat. No. 5,393,524). PFCs that aredisclosed as being useful for creating microbubbles includedodecafluoro-pentane (DDFP), sulfur hexafluoride, pentane,hexafluoropropylene, octafluoropropane, hexafluoroethane,octafluoro-2-butyne, hexafluorobuta-1,3-diene, isoprene,octafluorocyclobutane, decafluorobutane, cis-2-pentene, dimethylsulfide, ethylarsine, bromochlorofluoromethane, trans-2-pentene,2-chloropropane, hexafluorodisulfide, ethylmercaptan, diethylether,ethylvinylether, valylene, trisfluoroarsine, furfuyl bromide,cis-propenyl chloride, bytyl fluoride, 1,1 dichloroethane, isopropylmethyl ether, isopropylamine, methylfomate, 2-acetyl-furan,ethylenefluoride, 1-pentene, isopropylacetylene, perfluoropentane,isopentane, vinyl ether, 2-butyne, 1,4-pentadiene, tetramethyl silane,dimethyl phosphine, dibromodifluoromethane, 2-chloro-propene,difluroiodomethane, acetaldehyde, trimethyl boric, 3-methyl-2-butene,1,1 dimethylcyclopropane, aminoethane, vinyl bromide, disilanomethane,trichlorofluoromethane, bromofluoromethane, trifluorodichloroethane,perfluoropentene, and other fluorine containing hydrocarbons (U.S. Pat.No. 5,409,688).

Features which make the various species of microbubbles (e.g.,protein-coated, lipid-coated or surfactant-coated microbubbles, andmicrobubbles stabilized by gas that permeates very slowly across thebubble/liquid interface) useful as contrast media for ultrasound imagingare that such microbubbles can be prepared to a particular size range(e.g., 1μm to 6μm); and are stable in physiological solutions for atleast several minutes and up to several hours.

However, whether such microbubbles could be used as gas carriers, suchas to transport oxygen (O₂) to tissues, was not known or describedbefore the present invention. For example, for microbubbles to be usedto transport O₂, several practical and functional requirements must bedemonstrated: (1) the microbubbles must be of a sufficiently small sizeto pass through capillaries; (2) ordinary bubbles of such size dissolverapidly under the pressures of surface tension, and thus, themicrobubbles must be sufficiently stabilized to persist in thebloodstream; (3) the microbubbles must be permeable to oxygen and otherrespiratory gases; and (4) desirably, the microbubbles should unload O₂in tissues at high enough PO₂ to be physiologically or medicallyeffective. A potential disadvantage of using such microbubbles as gascarriers is the danger of embolization if infused microbubbles are, orbecome, too large in size.

SUMMARY OF THE INVENTION

The present invention relates to methods of using microbubbles as gascarriers to enhance delivery of one or more gases to or from bodytissues when such delivery is desirable. For examples, the gas carriersmay be used to deliver oxygen to body tissues which are deprived ofoxygen, to aid in supplying an anesthetic gas to tissue, to speedremoval of nitrogen or other inert gas from tissue to preventdecompression sickness in a diver or astronaut who will undergodecompression, to speed absorption of gaseous emboli by removing N₂ fromthe tissues, or to speed removal of toxic gas from body tissues. Themethods are especially useful for enhancing the transport of oxygen byone or more species of gas carriers for applications or conditions suchas substituting for deficiencies in hemoglobin function orconcentration, improving oxygen concentration in arterial blood in thecase of venous to arterial shunts, in right-to-left shunting, as well asfor organ perfusion, coronary angioplasty, oxygenation of ischemictissues resulting from vascular obstructions, and oxygenation of tissuesfor cancer radiation and chemotherapy.

In a preferred embodiment, the gas carriers comprise a specific type ofmicrobubble: a stabilized free gas microbubble, such as a microbubblecontaining a PFC. These stabilized free gas microbubbles have beenfound, unexpectedly, to carry significantly more oxygen to tissues thanan emulsion of liquid PFC of equivalent volume. Additionally, becausethe stabilized free gas microbubbles derive stability from a mechanismother than a surface coating which may act to some degree as apermeability barrier, the microbubbles have been found useful foroptimal transport of gases. In what follows, examples illustrate the useof microbubbles stabilized by free gas, such as by a PFC. Microbubblesstabilized by other mechanisms (e.g., protein-coated, lipid-coated orsurfactant-coated) which are small enough, permeable to the gas to becarried, and stable enough to persist in the body for at least 10 or 20minutes, are also considered for use in the methods according to thepresent invention. Similarity between PFC-stabilized microbubbles andmicrobubbles stabilized by other means, such as protein coatings orsurfactant monolayers at the surface, is disclosed herein (See also, VanLiew and Burkard, 1995, J Appl Physiol 79:1379-1385).

BRIEF DESCRIPTION OF THE DRAWINGS

(Some of the drawings have been reproduced, with permission, from theJournal Applied Physiology).

FIG. 1A is an illustration of the variations of blood pressure that amicrobubble encounters as it traverses the circulation. The horizontalaxis can be considered either as time spent in the circulation and itsvarious divisions, or as distance progressed through the circulation.Regions denoted are: V-systemic veins; PA-pulmonary artery; PB-pulmonarysmall-vessel bed; PV-pulmonary vein; A-arterial tree; SB-systemicsmall-vessel bed.

FIG. 1B is a graph representing variations of the volume in circulatinggas-stabilized microbubbles and of the constituent gases when a personbreathes 100 oxygen at normal pressure.

FIG. 2 is a graph representing variations of gas partial pressures incirculating gas-stabilized microbubbles where the breathing gas is 100%O₂.

FIG. 3 is a graphic representation of variations of gas partialpressures that a microbubble encounters as it traverses the circulationwhen the breathing gas is air.

FIG. 4 is a diagram showing absorptive pressures (ΔP_(abs)) vs. radiusfor a microbubble which contains a slowly-permeating gas, wherein Pγ ispressure due to surface tension, P_(win) is the partial pressure deficitin tissue caused by metabolism of O₂ (the O₂ window), and P_(bubX) ispartial pressure of the slowly-permeating gas inside the microbubble.

FIG. 5 is a graphic representation showing volume of a microbubble, andits constituent gases, that is circulating in the blood in anair-breathing person.

FIG. 6 is a graphic representation showing partial pressures of the twomajor gases, X and N₂, in the microbubble throughout the circulation inan air-breathing person.

FIG. 7 is a graphic representation showing radius of a stabilizedmicrobubble as it circulates in the blood.

FIG. 8 is a graphic representation showing the O₂ content of a single,gas-stabilized microbubble as a function the P_(O2) in the surroundingblood.

FIG. 9 is a graphic representation showing alterations (partial pressureof the stabilizing gas, radius, and volume) of a single, gas-stabilizedmicrobubble as a function the P_(O2) in the surrounding blood.

FIG. 10 is a graphic representation showing dependence of microbubble O₂content on alveolar P_(O2) (open circles) under hyperbaric conditions.

FIG. 11 is a graphic representation showing dependence of microbubble O₂content (solid) and radius (dotted) on P_(O2) in the surroundingenvironment for differing amounts of gas X in the microbubble.

FIG. 12 is a graphic representation illustrating the curves for O₂content of blood alone, microbubbles alone, and a combination of bloodand microbubbles (the microbubble concentration is 4.7×10⁸ /ml).

FIG. 13 is a graphic representation illustrating the curves for O₂content of blood alone, microbubbles alone, and a combination of bloodand microbubbles (the microbubble concentration is 1.8×10⁹ /ml).

FIG. 14 is a graphic representation showing CO₂ content of a single,gas-stabilized microbubble as a function the P_(O2) in the surroundingblood and tissue locations.

FIG. 15 is a graphic representation showing preparation of aan animalfor monitoring physiologic values during microbubble treatment.

FIG. 16 is a graph showing variations of muscle PO₂ after infusion ofstabilized microbubbles.

FIG. 17 is a graphic representation showing variations of gas volumes ina microbubble in a person who breathes 100% O₂, but has N₂ in thetissues.

FIG. 18 is a schematic representation of an instrumented pig.

FIG. 19 is a graph showing the time course of arterial oxygen tension(PaO₂) during application of right to left circulatory shunt in thelungs, inhalation of oxygen and microbubbles infusion.

FIG. 20 is a graph showing the time course of oxygen tension inabdominal muscle (PO₂) measured with Kontron transcutaneous PO₂₋₋ PCO₂sensor during the application of right to left shunt, inhalation ofoxygen and microbubbles infusion.

FIG. 21 is a graph showing the time course of oxygen tension inabdominal muscle (PO₂) measured with Radiometer transcutaneous PO₂₋₋PCO₂ sensor during the application of right to left shunt, inhalation ofoxygen and microbubbles infusion.

FIG. 22 is a graph showing the time course of carbon dioxide tension inarterial blood (PaCO₂) during the application of right to left shunt,inhalation of oxygen and stabilized microbubbles infusion.

FIG. 23 is a graph showing the time course of carbon dioxide tension inabdominal muscle (PCO₂) measured with Kontron transcutaneous PO₂₋₋ PCO₂sensor during the application of right to left shunt, inhalation ofoxygen and stabilized microbubbles infusion.

FIG. 24 is a graph showing the time course of carbon dioxide tension inabdominal muscle (PCO₂) measured with Radiometer transcutaneous PO₂-PCO₂ sensor during the application of right to left shunt, inhalationof oxygen and stabilized microbubbles infusion.

FIG. 25 is a representation of the effects of stabilized microbubblesinfusion on oxygen tension in arterial blood during right to leftcirculatory shunt in the lungs of Group 2 animals.

FIG. 26 is a representation of oxygen tension in arterial blood duringright to left circulatory shunt in the lung treated with stabilizedmicrobubbles and oxygen breathing in seven animals.

DETAILED DESCRIPTION OF THE INVENTION

By the term "right to left circulatory shunt" for the purposes ofspecification and claims is meant a condition wherein deoxygenatedvenous blood for whatever reason is not subject to reoxygenation in thelungs before making its way to the left heart and the systemiccirculation.

In a preferred embodiment of the methods of the present invention, aspecific type of gas carrier is utilized for optimal transport of gasesto or from tissue. Particularly, the gas carrier comprises microbubblesformed by suspending, in a liquid, a quantity of gas, preferably gaseousPFC. Thus, microbubbles are formed which contain a foreign gas that,when introduced into the bloodstream, permeates through themicrobubble/blood interface very slowly. The slowly permeating gasserves as a stabilizer of the microbubble structure. It will beappreciated by those skilled in the art that the size of themicrobubbles formed can be controlled by the manufacturing process to besufficiently small so as not to obstruct the systemic or pulmonarycapillaries. Particularly useful in producing such microbubbles arecompounds comprising gases and PFCs which are a liquid at temperaturesof manufacturing the compound, but become a vaporized gas at bodytemperature thereby forming microbubbles (such compounds are availablefrom SONUS Pharmaceuticals). The gases and PFCs useful in the productionof such microbubbles are disclosed in, for example, U.S. Pat. Nos.5,393,524, and 5,409,688 (also in U.S. Ser. Nos. 08/380,085, 08/008,172,08/148,284, and 08/182,024) all of which are incorporated herein byreference. Thus, while other such compounds are useful for methodsaccording to the present invention, for purposes of illustration, butnot limitation, microbubbles used in the examples compriseperfluoropentane, and more particularly contain the PFCdodecafluoropentane (DDFP). The microbubbles are prepared by aphase-shift technology (See, for example, U.S. Pat. No. 5,393,524),whereby an emulsion of liquid DDFP droplets is prepared in a coolenvironment, and then when infused or injected into the body of anindividual, the droplets become gas microbubbles. Depending on theparticular compound, the microbubbles are stabilized to laste in thebloodstream for a time ranging from a few minutes to several hours. Thefollowing examples illustrate novel properties of these microbubbles andother stabilized microbubbles, and parameters to consider relating totheir use for the methods of the present invention.

EXAMPLE 1 Oxygen Transport By Stabilized Microbubbles

The mechanisms involved in O₂ transport by stabilized gas microbubblescan be illustrated using equations based on physical principles.Equation 1 is based on the equality of hydrostatic pressures on amicrobubble (lefthand side of the equation) with the sum of partialpressures of the gases inside (righthand side of the equation) where: Xrepresents the gas that permeates slowly from the microbubble; P_(B) isbarometric pressure; γ is surface tension; R is microbubble radius; Pb1is blood pressure; and Pbub is the partial pressure of the respectivegas inside the microbubble.

    Equation 1: P.sub.B +2γ/R+Pb1=Pbub.sub.N2 +Pbub.sub.O2 +Pbub.sub.CO2 +Pbub.sub.H2O +Pbub.sub.X

Equation 2 shows that when gas X does not permeate the microbubble/bloodinterface, the partial pressure of gas X will increase or decrease whenparameters (righthand side of the equation) change, and shows thatpressure due to surface tension is inversely proportional to microbubbleradius. Blood pressure and partial pressures, in blood, of O₂ and CO₂(Pb1_(O2) and Pb1_(CO2)) differ in parts of the circulatory system, andarterial partial pressures of O₂ and CO₂ (Pa_(O2) and Pa_(CO2)) dependon breathing gas and respiratory pattern. Note that immediately afterblood has gone through the lungs, Pb1_(O2) equals Pa_(O2) and Pb1_(CO2)equals Pa_(CO2). Derivation of Equation 2 was previously disclosed(Burkard and Van Liew, 1994, J Appl Physiol 77:2874-2878).

    Equation 2: Pbub.sub.X =2γ/R+Pb1+(Pa.sub.O2 -Pb1.sub.O2)+(Pa.sub.CO2 -Pb1.sub.CO2)

Equation 3, derived from Boyle's law, shows that radius, R, isdetermined by the ratio of the unchanging volume of gas X at standardpressure (V_(X),8) where Ps is standard pressure, to the markedlyvariable partial pressure of gas X.

    Equation 3: R=[3Ps V.sub.X,8 /4πPbub.sub.X ].sup.1/3

Thus, when Pbub_(x) changes in different parts of the circulation, theradius will change and the volumes of permeant gases, such as O₂, mustchange in accordance with their near partial-pressure equilibriumbetween inside and outside of the microbubble.

These dynamics can be illustrated with an equation system describedpreviously (Burkard and Van Liew, 1994, Respir. Physiol. 95:131-145;herein incorporated by reference). The system takes into considerationthe major phenomena that determine growth and absorption ofmicrobubbles, including diffusion of any number of gases across themicrobubble/blood interface, surface tension, the lowering of P_(O2) byutilization of O₂ by tissues (known as the O₂ window) , and Boyle's lawfor pressure dependence of gas volumes. Additionally considered weremicrobubble size and composition as a function of time and location inthe circulatory system, including the effect of blood pressure and itschanges with the pumping action of the heart.

Oxygen Carrying Capacity

FIG. 1A shows the variations of blood pressure that a microbubbleencounters as it traverses the circulation. FIG. 1B shows changes of theamount of O₂ (V_(O2)) in a microbubble when it moves through thecirculation. In a person who breathes pure O₂ during administration ofthe gas carrier (microbubbles), O₂ is approximately 70% of their volume(FIG. 1B). Thus, using the values from FIG. 1B as an example, 0.11 pl ofO₂ (volume of O₂ in picoliters) was carried in a microbubble that had avolume in the lungs (regions PB and PV, for pulmonary small-vessel bedand pulmonary veins) of 0.125 pl (0.11/0.125=90%) and a diameter of 6μm. At 0.11 pl O₂, /microbubble, transport of 5 vol % of O₂ requires4.6×10⁸ microbubbles/ml, and the volume of gas would be 0.07 ml, i.e. 7ml gas/100 ml blood in the lung, and 0.02 ml in the systemicsmall-vessel bed.

Based on further studies, shown in Table 1 is a comparison of estimatesfor the capacity for O₂ transport between microbubbles comprised of DDFPemulsion, emulsions of liquid perfluorooctyl bromide (PFOB), and bloodand plasma, for a person breathing pure O₂ at normal pressure. PFOB is aPFC emulsion, used clinically for O₂ transport, having one of thehighest solubilities for O₂, (Lowe, 1991, Chem. Ind. Lond. 3:83-89). Ascan be seen by FIG. 1, and the comparison in Table 1, microbubbles cancarry approximately 90 ml of useable O₂ (standard pressure) per 100 mlof microbubble volume (ambient pressure); this is almost twice the O₂carrying capacity of the same volume of PFOB, and four times thecapacity of whole blood. The results of the comparisons showed animprovement in O₂ carrying capacity much greater than expected. Also, arelatively small amount of the PFC microbubbles is needed, as comparedto PFOB, to attain a given O₂ delivery.

                  TABLE 1                                                         ______________________________________                                                      O.sub.2 capacity                                                                           Circulating Foreign                                  O.sub.2 carrier (by volume) Liquid*                                         ______________________________________                                        microbubbles/DDFP                                                                           90 ml O.sub.2 /100 ml                                                                      0.9 ml cf DDFP**                                      gas                                                                          PFC emulsion 50 ml O.sub.2 /100 ml 600 ml of PFOB                             (PFOB) PFOB                                                                   Whole blood 22 ml O.sub.2 /100 ml 0                                            blood                                                                        Plasma 2.3 ml O.sub.2 /100 ml 0                                                blood                                                                      ______________________________________                                         *for delivery of 5 vol % of O.sub.2                                           **approximately 135 ml of gaseous DDFP                                   

Unloading of O₂

FIG. 2 shows the partial pressures of gases in microbubbles and bloodfor a person breathing 100% O₂. Blood and microbubble P_(O2) are sonearly equilibrated that one curve suffices for both; both rise as theblood exchanges gas in the pulmonary small-vessel bed, and fall duringexchange with tissues in the systemic small-vessel bed. Pbub_(x) risesin the pulmonary artery due to pulmonary arterial pressure, and falls inthe pulmonary small-vessel bed because blood pressure is less andbecause of exchange of O₂. Pbub_(x) rises again in systemic arteries dueto high blood pressure, and then rises in the peripheral small-vesselbed because of the loss of O₂ in that region. As shown in FIG. 2 andFIG. 1B, respectively, there is a release of O₂ by the fall of P_(O2)but there is also additional release due to decrease of microbubblevolume. Therefore, microbubbles deliver O₂ at a higher PO₂ than from aliquid containing physically-dissolved O₂, such as a liquid PFC, whereinthe P_(O2) fall is directly proportional to the amount extracted.

EXAMPLE 2 Stability and Function in Blood Circulation

By using physical gas-law equations, it can be shown that microbubblesstabilized by a slowly diffusing gas can change size as they movethrough the circulatory system. Microbubble changes encountered inultrasonic imaging have been described previously (Van Liew and Burkard,1995, Invest Radiol 30:315-321). Equation 4 shows that in a givenenvironment with given Pbub_(x), total volume of the microbubble dependson the amount of gas X present:

    Equation 4: Pbub.sub.x =Ps V.sub.X,8 /V.sub.T w

herein Ps is standard pressure; V_(X),8 is volume of gas X in amicrobubble at body temperature, standard pressure; and V_(T) is volumeof a microbubble at ambient temperatures.

Equation 5, a rearrangement of Equation 3, shows that for a given amountof gas X, Pbub_(x) is determined by microbubble size; since radius, R,changes when other gases enter or leave the microbubble; Pbub_(x) willalso change.

    Equation 5: Pbub.sub.x =3Ps V.sub.x,s /4πR.sup.3 =C.sub.rx /R.sup.3

wherein C_(rx) is a constant equal to 3Ps V_(x),s /4 π.

Variations of blood pressure and gas concentrations

FIG. 3 shows the partial pressures of gases in microbubbles and bloodfor a person breathing air. In FIG. 3, the P_(O2) rises from its lowvalue in the venous side of the circulation (SV=systemic vein;PA=pulmonary artery) to the alveolar level in the pulmonary small-vesselbed (PB) and falls when the microbubble reaches the systemicsmall-vessel bed (SB, in the tissues). The P_(CO2) changes only slightlythrough the circulation. The sum of the partial pressures of alldissolved gases (ΣPbl_(i)) is subatmospheric by an amount equal to thepartial pressure deficit due to O₂ metabolism (the O₂ window) in venousblood, but is equal to atmospheric pressure in arterial blood.

The response of a particular microbubble to the circulation environmentas depicted in FIG. 3 will depend on its stabilization mechanism. FIG. 4presents the characteristic diagram for a microbubble which contains aslowly-permeating gas, as is a preferred embodiment in the methods ofthe present invention. Note that the curve for partial pressure of gas Xin the microbubble (Pbub_(x)) shows a large negative ΔP_(abs) (a growthpressure) at small radii. Partial pressure of gas X exactly balances thepressure due to surface tension at the stable radius (circle). If themicrobubble should have a radius that is not at the stable radius,partial pressure of gas X would be out of balance with surface tensionpressure, so the microbubble would either grow (if the radius were belowthe stable radius) or shrink if the radius were above the stableradius). The Pbub_(x) curve was drawn from Equation 5 using enough gas Xto give stable radii of 1.85 μm in the systemic vein (lower circle), and2.0 μm in the pulmonary vein (higher circle) when the person breathesair. In arteries, blood pressure would add an additional absorptivepressure which is independent of radius, so a second horizontal line, inaddition to the O₂ window line, would be needed. The function ofstabilizing mechanisms has been outlined previously (Van Liew andBurkard, 1995, J Appl Physiol 79:1379-1385).

To demonstrate how the changing conditions depicted in FIG. 3 affect amicrobubble with the characteristics depicted in FIG. 4, simulations ofthe course of microbubble size and composition as functions of time andlocation in the circulatory system were performed using thenumerically-solved system of equations (Burkard and Van Liew, 1994,supra). The equation system accounts for a "gradient region" immediatelyoutside the microbubble with thickness which is proportional tomicrobubble radius. In the gradient region, the partial pressures ofgases decline or rise between the levels in the microbubble and in theblood far away. The equation system incorporates the assumption thatthere is a stagnant layer of blood around the microbubble which isthicker than the gradient region; for microbubbles as small as the oneswe are concerned with as being useful in the methods of the presentinvention, diffusive exchanges are little affected by convection outsidethe microbubble and disregard of convection effects gives conservativeestimates of rates of equilibration between microbubbles and thesurroundings. Variables in the equation system are time; radius of themicrobubble; area of the microbubble; volume of the microbubble understandard conditions; volume of the microbubble under ambient conditions;and partial pressures of all gases that are under consideration 1) inblood far from the microbubble, 2) in the gradient region in bloodaround the microbubble, and 3) inside the microbubble. Parameters andtheir values are listed in Table 2.

                  TABLE 2                                                         ______________________________________                                                     Solubilities  Diffusivities                                        Respiratory Gas ml × ml.sup.-1 100 Kpa.sup.-1 cm.sup.2 /min           ______________________________________                                        N.sub.       0.0146        1.32 × 10.sup.-3                               O.sub.2 0.0227 1.24 × 10.sup.-3                                         CO.sub.2 2.35 1.05 × 10.sup.-3                                        ______________________________________                                    

Surface tension of blood=50 dyn/cm

To consider the effects of the circulation environment as depicted inFIG. 3, the microbubble was assumed to contain a slowly permeating gas(gas X, Equation 5), as well as the O₂, CO₂, and N₂ that are normallyfound in the body. Diffusion of gas between microbubbles and blood wasassumed to have no effect on the gas concentrations in the blood beyondthe gradient region, as if the microbubbles are isolated from eachother.

The top trace of FIG. 5 shows, for an air-breathing person, in situvolume of a circulating microbubble that is stabilized by a slowlypermeating gas, as characterized by FIG. 4. The 1.85 μm stable radiusthat was seen in FIG. 4 gives a stable volume of 0.027 picoliters in thesystemic veins (SV, FIG. 5). As shown in FIG. 5, in the pulmonary artery(PA) , the total volume (V_(T)) shrinks slightly and the curve hasripples that reflect the systolic and diastolic blood pressures. In thepulmonary small-vessel bed (PB), blood pressure decreases and P_(O2)increases, so the microbubble grows to reach 0.034 picoliters. In thesystemic artery (SA), where blood pressure is substantial, the volumeshrinks. In the tissue small-vessel bed (SB), blood pressure decreasesat the same time as the P_(O2) decreases; the net effect is an increasein mean volume.

In situ volume (expressed at ambient conditions) varies less for gas Xthan for other gases because gas X, to be useful in the method of thepresent invention, has a very low permeation coefficient. The changes ofhydrostatic pressures affect volume of a constant number of molecules ofgas X by Boyle's Law, whereas the other gases gain or lose molecules dueto diffusion as well as responding to hydrostatic pressure changes. TheO₂ and N₂ volume traces show that the major cause of growth of totalmicrobubble volume in the lungs is diffusive entrance of O₂ and N₂ intothe microbubble. The microbubble releases these gases in tissue so thereis net transport from lung to tissue.

FIG. 6 shows partial pressures of the two major gases, X and N₂, in themicrobubble throughout the circulation in an air-breathing person. Thespikes and ripples on the upper trace show brief periods when Pbub_(N2)(partial pressure of N₂ gas in the microbubble) and Pb1_(N2) (partialpressure of N₂ gas in the blood) are not in equilibrium. Diffusion of N₂is so rapid, compared to diffusion of gas X, that PN₂ in the microbubbleremains close to PN₂ in the surroundings while the microbubble undergoesthe large changes in N₂ volume seen in FIG. 5. In contrast, Pbub_(x)varies with location due to hydrostatic pressure changes and dilution orconcentration of gas X caused by the exchanges of other gases.

The Pbub_(x) trace in FIG. 6 is inversely related to the total volumetrace of FIG. 5 and the radius trace of FIG. 7, as expected fromEquation 5. Because of the cubic relation between radius and volume,microbubble radius undergoes much smaller changes than volume does. Inthe systemic vein (SV), Pbub_(x) is about 60 kPa (FIG. 6). This pressurecan be seen in FIG. 4 directly below the left stabilized-radius circle;and it counters the absorptive pressures of Pγ (pressure due to surfacetension) of about 50 kPa and the O₂ window (inherent unsaturation due tostable metabolism) of about 7 kPa. According to FIG. 4, the radius is1.85 μm in the systemic vein and 2.0 in the pulmonary artery; theseradii are to be seen on the curve of FIG. 7. Except for the slowlypermeating gas, the contents of the microbubble are essentiallyequilibrated with their surroundings immediately after progressing fromone part of the circulation to another. Of particular note, not only dothe microbubbles stabilized by a slowly permeating gas depicted in FIG.5 have a size which is similar to size of erythrocytes, but they alsoshare diffusion characteristics and times to equilibration.

Microbubble Size

Microbubbles smaller than 3 μm radius probably pass through thecapillary beds easily but larger microbubbles may become lodged.Microbubbles with radii slightly larger than capillaries may also beable to pass through due to the push of blood pressure. Experimentsindicated that the cutoff radius for passage through the lungs is below11μ in dogs (Butler et al., 1979, J. App. Physiol. 47:537-543). FIG. 7shows that microbubbles approach maximal radius in the pulmonarysmall-vessel bed, so microbubbles that can traverse other parts of thecirculation may lodge in pulmonary vessels. This is especially so if themicrobubble grows upstream of smaller vessels, as would occur if theexchanges of O₂, CO₂, and N₂ occur in pulmonary arterioles. Microbubblesmay dislodge if loss of gaseous contents by outward diffusion makes themsmall enough. The range of diameter of microbubbles stabilized by aslowly permeating gas would have a lower end of approximately 1 μm, witha limiting factor being the ability to withstand the various pressuresencountered in the blood while maintaining diffusivity; and a high endof approximately 6 μm, with a limiting factor being the ability to passthrough capillaries without becoming temporarily or permanently lodgedtherein.

Diffusivity Relative To Microbubble Size and Lifespan

Rate of exit of the slowly permeating gas from a gas-stabilizedmicrobubble determines how long the microbubble lasts. Thus,microbubbles which contain a slowly permeating gas X can be expected topersist in the circulation until all of gas X has diffused out into theblood and from there out into the atmosphere via lung exchange. It hasbeen reported that microbubbles of DDFP yield observable ultrasonicsignals for 12 minutes after intravenous injection (Kenny et al. 1994, JAm Coll Cardiol, 21:450A); and that the microbubbles may persist 12additional minutes after they are too small to be detectedultrasonically. Methods to identify those gases (slowly permeating gasX) which may be used to stabilize a microbubble are described in U.S.Pat. No. 5,409,688. In a preferred embodiment, gas X has a low watersolubility (e.g., less than 10 micromoles/liter), a high density (e.g.,greater than 10 kg/m³), a large molar volume (e.g., greater than 100 cm³/mole), or permeates across the microbubble/blood interface more slowlythan N₂, or a combination thereof. The slowly permeating gas stabilizesthe microbubble by diluting the O₂, CO₂, and/or any other gases in themicrobubble, thereby lowering their partial pressures. Therefore, whenthe microbubble is in an equilibrium state for a given environment, thepartial pressures of permeant gases can be the same inside and outsideeven though the total pressure inside the microbubble may be above thesum of partial pressures in the blood because of surface tension andblood pressure. Such microbubbles are "stabilized", meaning that themicrobubbles stabilized by a slowly permeating gas are absorbed moreslowly than air bubbles.

Alternative Stabilizers

Microbubbles that are stabilized by other means, different fromstabilization by slowly permeating gas, can also serve to carry gas toor from tissue. To understand the function of other stabilizingmechanisms, such as albumin coatings, surfactant monolayers, orsemi-solid coatings, consider that the roles played by gas X arefulfilled by the alternative stabilizer. In particular, pressure exertedby gas X in FIGS. 2, 4, 6, and 9 is exerted instead by the alternativestabilizing mechanism. Comparison of stabilization by a slowlypermeating gas, such as a PFC gas, with stabilization by alternativestabilizing mechanisms has been described previously (Van Liew andBurkard, 1995, J Appl Physiol 79:1379-1385).

EXAMPLE 3 Stabilized Microbubbles as Oxygen Transporting Agents in Blood

As described herein, stabilized microbubbles can pass through capillarybeds, recirculate for between a few minutes to hours, and carry O₂ fromthe lungs to the tissues. To appreciate the clinical usefulness of suchstabilized bubbles for O₂ transport in blood, it is necessary tounderstand the relation-ship between the O₂ content of a stabilizedmicrobubble and the P_(O2) in the blood surrounding the microbubble.Further, for clinical applications, it is necessary to understand thealterations of the microbubbles during O₂ transport. As describedherein, high P_(O2) in the lungs causes O₂ to diffuse into themicrobubble resulting in (a) a microbubble with a larger volume; and (b)an initially lower P_(CO2) inside the microbubble thereby causing CO₂ todiffuse into the microbubble. Thus, compared to microbubbles in veins,microbubbles leaving the lung contain considerably more O₂ and slightlymore CO₂, thereby transporting these gases from lung to tissues. Becausethe microbubbles contain gases that can diffuse in and out of themicrobubbles easily, the microbubbles undergo much larger size changesin the blood than a fluid-filled blood cell would undergo. Whereas aliquid would deliver O₂ only in physical solution in proportion to afall of PO₂, the microbubbles release more O₂ because there is a volumechange in addition to a drop of P_(O2).

To understand the relationship between the bubble content of _(O2)(Pbub_(O2)) and the various partial pressures of O₂ dissolved in theblood throughout circulation (Pbl_(O2)), it is necessary to express thecontent of O₂ in terms of microbubble radius and P_(O2) (Equation 6) andto relate the microbubble radius to environmental influences in thecirculation (Equation 7). Such influences include physical forces andthe gas partial pressures which exist in the blood at a particularlocation in the circulation.

Equation 6 ##EQU1##

According to Equation 6, moles of O₂ contained in a stabilizedmicrobubble is a function of Pbub_(O2) and the cube of radius (R)(volume). In the stable state, partial pressures of readily-permeablegases are equal inside and outside, so stable radius (R*) is a functionof Pbl_(O2) at any particular location in the circulatory system.Relevant to O₂ transport by the microbubble in the circulation, and asevident in Equation 6, both Pbub_(O2) and R* increase when themicrobubble takes on O₂, and decrease when the microbubble unloads O₂.The change of microbubble volume makes the O₂ unload at considerablyhigher P_(O2) than would be the case if only P_(O2) changed.

Equation 7 ##EQU2##

Thus, the stable radius for a microbubble at an arbitrary location inthe circulatory system is a function of local blood pressure(P_(blood))) and P_(O2) and P_(CO2) in the blood at that location.Equation 7 is a cubic polynomial which can be solved for R* bysuccessive approximations. From Equation 7, the radius at a particularPbl_(O2) can be obtained and then used in Equation 6.

FIG. 8 shows the O₂ content of stabilized microbubbles relative to theP_(O2) of the blood. The diagonal line is the O₂ -content-vs.-P_(O2)shape for an O₂ carrier that changes P_(O2) without changing volume. Aliquid which contained the amount of physically-dissolved O₂ shown bythe open circle would unload O₂ along the diagonal line. At 60 kPa, theO₂ unloaded by volume change alone (open arrow) is greater than thatunloaded by P_(O2) change alone (filled arrow). The curvature of thecontent-P_(O2) curve depends on the size of the microbubble and thearterial P_(O2). For very small microbubbles, there is less curvature;for a microbubble with 0.5 μm radius, the content-P_(O2) curve wouldalmost superimpose on the diagonal in FIG. 8.

FIG. 9 shows the alterations of the microbubbles in a person breathing100% O₂ as a consequence of circulating in the blood where P_(O2) variesdepending on the location. For example, total volume of the microbubblequadruples between tissue capillaries (left side of FIG. 9) and thepulmonary vein (right side of FIG. 9). Note that there is volume, due togas X, CO₂ and H₂ O vapor, in the microbubble when the P_(O2) is zero.The change of radius is less marked than the change of volume because ofthe cubic relation between volume and radius of a sphere. FIG. 9 alsoshows that the pressure contributed by stabilizing gas X changes from alittle over 30 kPa, when the microbubble is full of O₂, to 140 kPa whenthere is no O₂.

To further understand the relationship between the O₂ content and thealveolar P_(O2) (P_(AO2)), illustrated in FIG. 10 are examples whichshow the amount of O₂ in a microbubble of given radius to be directlyproportional to the alveolar P_(O2), consonant with Equation 6. Alsoshown, is that increased amounts of O₂, can be carried in hyperbaricconditions. Using 3 μm as the micro-bubble radius for the arterial pointfor these three curves, in situ volume is the same for the threeexamples; but the O₂ content is expressed at standard pressure. In theright-hand curve of FIG. 10, applicable for a person with an ideal lungwho is breathing O₂ at 3 atm abs, the microbubble carries more than 3times as much O₂ as when the person breathes pure O₂ at normal pressure(left-most curve). The results of such a comparison show an improvementin O₂ carrying capacity much greater than expected. Perhaps moreimportant, the curvature of the 289 kPa trace is much greater than whenP_(AO2) is less; i.e., the unloading P_(O2) is very high, with half theO₂ unloaded when P_(O2) is about 270 kPa, only 20 kPa below the alveolarlevel.

Microbubble size at any P_(O2) is determined by C_(X), wherein C_(X) isdirectly proportional to the volume of gas X in the microbubble (i.e.,R³ =C_(X) /Pbub_(X)). FIG. 11 illustrates how three different values ofC_(X) give different O₂ contents and different radii. Radius is themanifestation of a microbubble that one would observe with a microscope.Moderate changes of the observed radius are associated with largechanges of O₂ volume in a microbubble. For example, a microbubble in thealveolar capillaries, with a radius of 1 μm (C_(X=) 92 kPa-μm³) has anO₂ content that is about 2% that for a microbubble having a radius of 3μm (C_(X=) 920 kPa-pm³).

To appreciate carriage of O₂ by individual microbubbles stabilized byslowly permeating gas, it is important to understand functionalproperties of such microbubbles in the circulation, and methods of usingsuch microbubbles for clinical applications according to the presentinvention should consider the effect of introducing a suspension of themicrobubbles into whole blood. The combination of normal blood (normalerythrocyte count is 5×10⁹ cells/ml) with 4.1×10⁸ microbubbles/ml(maximal size of R* of the microbubbles is 3 μm) is shown in FIG. 12.This number of microbubbles raises arterial O₂ content 5 vol % above thenormal value (line marked "Total"), which is the normal arteriovenous O₂content difference. Thus the O₂ for normal metabolism could be suppliedby the microbubbles. According to the example illustrated in FIG. 12,about a quarter of the O₂ in the blood-microbubble mixture would beunloaded at a P_(O2) above 20 kPa so the arterial end, and perhaps theentire extent, of tissue exchange units (arterioles and capillaries)would have high P_(O2). FIG. 13 shows the blood O₂ content-P_(O2) curvefor a combination of blood plus 1.8×10⁹ microbubbles/ml results in adoubling of the O₂ capacity. Thus, half the combination's _(O2) would beunloaded at high P_(O2) above the steep part of the O₂ -Hb curve.

Carriage of CO₂ From Lungs to Tissue

The loading and unloading of O₂ into the microbubble drives changes ofthe microbubble's CO₂ content. FIG. 14 illustrates CO₂ carriage bymicrobubbles stabilized by slowly permeating gas. The open circle on thecurve for P_(CO2=) 5.2 kPa (40 mmHg) represents the arterial point in anO₂ -breathing person. For a given P_(CO2), about 0.0045 picoliters ofCO₂ would be unloaded in tissue as P_(O2) goes from the arterial levelto zero. The two filled circles at the left are hypothetical venouspoints assuming that venous P_(O2) =5 kPa; if P_(CO2) =6.8 kPa, about0.0002 picoliters less CO₂ is unloaded in tissue than if P_(CO2) =6.0kPa. Accordingly, the amounts of CO₂ carried by microbubbles for anarteriovenous P_(CO2) of 0.8 kPa (6 mmHg) and venous P_(O2) of 5 kPa forbreathing of air (_(O2) at 1 atm abs, and O₂ at 3 atm abs) were 0.001,0.004, and 0.005 picoliters per microbubble, and 5%, 4% and 2% of thevolume of O₂ carried, respectively.

Thus, as a consequence of O₂ carriage from lungs to tissue bymicrobubbles, CO₂ is carried also, in the direction counter to the usualcarriage from tissues to lungs. If microbubbles are used to carry 5 vol% of O₂ in a person breathing pure O₂ at normal pressure, estimatesindicate that the magnitude of reverse carriage of CO₂ would be about0.2 volt. A crude estimate indicates that the body would come to a newsteady state having average tissue P_(CO2) elevated by about 0.03 kPa(less than 0.25 mmHg) above the normal value near 5 kPa. If 20 volt ofO₂ is carried, tissue P_(CO2) might be elevated by 0.1 kPa (less than 1mmHg). Thus the effect of the small retrograde carriage of CO₂ isnegligible.

EXAMPLE 4 Methods of Using Stabilized Microbubbles as O₂ Carrier

4.1 Method for delivering 0, to tissues in place of blood.

Stabilized microbubbles may be used in a method of therapeuticallytreating an individual in need of a blood transfusion, i.e., forimproving delivery of oxygen to oxygen-depleted tissues; or forreversing effects of lack of functional hemoglobin such as caused byblood loss, anemia, carbon monoxide poisoning, or other causes.According to this method of the present invention, atherapeutically-effective amount of microbubbles is introduced into theblood circulation of an individual, wherein the microbubbles function tocarry and allow exchange of metabolic gases in adequate amounts and atadequate pressures for sustaining the respiratory metabolism of thetreated individual. One skilled in the art would appreciate that theamount of microbubbles to be introduced (number of microbubbles/ml)depends on a number of factors including the particular stabilizingmechanism used for microbubble preparation, the size (radius) range ofthe microbubbles in the preparation, the volume of blood in theindividual to be treated, and the extent of oxygen depletion in theindividual to be treated. The therapeutically effective amount ofmicrobubbles can be administered into the blood circulation of theindividual to be treated by methods known in the art, includingintravenous administration.

For example, the therapeutically effective amount of microbubbles can beadministered by injection or infusion into a vein of the individual tobe treated who is undergoing oxygen inhalation; and metabolic gases aremonitored. The therapeutically effective amount of the microbubbles maybe administered by a single injection, multiple injections, or aninfusion until stable and medically acceptable levels of P_(O2) havebeen attained. Using this method with a person with a severeright-to-left shunt (50% shunt), for example, atherapeutically-effective amount of a microbubble preparationadministered to a microbubble-to-blood volume ratio of approximately 5%may increase arterial P_(O2) from about 45 mmHg to about 60 mmHg whenthe individual inhales a gas mixture with 60% oxygen. Increased oxygentransport could be attained in the method according to the presentinvention by increasing the amount of oxygen that the treated individualinhales (e.g. 100% or pure O₂, or using hyperbaric conditions).Advantages of using DDFP microbubbles in a method of therapeuticallytreating an individual having oxygen-depleted tissues include: (a)significantly more oxygen can be carried to the tissues than a PFCemulsion of equivalent volume; and (b) as a result of (a), the amount offluorocarbon that has to be injected to produce a given volume ofmicrobubbles is almost 2 orders of magnitude less than the equivalentvolume of liquid PFC emulsions, thereby resulting in a decrease offoreign matter introduced into an individual to be treated. However,care must be taken to avoid or ameliorate oxygen toxicity caused by ahigh P_(O2). References which demonstrate the usefulness of liquid PFCemulsions to transport oxygen to oxygen-depleted tissues include Zuckand Riess, 1994, Crit. Rev. Clin. Lab. Sci. 31:295-324; Geyer, 1988,Biomater. Artif. Cells Artif. Organs 16:31-49; Mitsuno et al., 1984,Artif. Organs 8:25-33; Faithfull, 1992, Biomater. Artif. CellsImmobilization Biotechnol. 20:797-804; Goodin et al., 1994, Crit. CareMed. 22:680-689; Lowe, 1987, Comp. Biochem. Physiol. A. 87:825-38;Millard, 1994, Artif. Cells Blood Substit. Immobil. Biotechnol.22:235-244; Lowe, 1991, Vox Sang 60:129-140; and Johnson et al. 1994, JAppl Physiol 79:1777-1786.

4.2 Method for delivering O₂ to ischemic tissues.

Ischemia occurs in an organ or tissue (individually or collectivelyreferred to as "organ") deprived of blood flow, whether cessation ofblood circulation through the organ is, for example, a result ofstopping the heart beat for surgical procedures; or because of naturalcauses such as in a heart attack. Depending upon the extent and durationof the lack of oxygen during the ischemic process, cell swelling canoccur, along with loss of normal cellular integrity eventually leadingto cell death.

Thus, for example, an aduring thupply of oxygen is needed during theperfusion process and/or resuscitation process (individually orcollectively referred to as "perfusion") to minimize ischemic damage (oravoid further ischemic damage) in organs intended for transplantation.In one mode of this embodiment, and using methods and compositionsoutlined in Examples 1-4 herein, an organ is perfused. The methodcomprises perfusing the organ with a buffered oxygenated physiologicalsolution containing a therapeutically effective amount of the stabilizedmicrobubble preparation to flush the organ to remove blood and acidoticproducts which have accumulated in the organ during blood flowdeprivation, and to provide for adequate oxygen delivery to the ischemicorgan. References which demonstrate the usefulness of liquid PFCemulsions in perfusion of organs include Geyer et al. 1988, supra;Faithfull, 1992, supra; Millard, 1994, supra; Kloner and Hale, 1994,Artif. Cells Blood Substit. Inunobil. Biotechnol. 22:1069-1081; Martinet al., 1993, Ann Thorac Surg 55:954-60; Kuroda et al., 1991,Transplantation 52:989-91; and Bando et al., 1989, J Thorac CardiovascSurg 98:137-45.

Likewise, catheter balloon inflation performed during percutaneoustransluminal coronary angioplasty results in a temporary interruption ofcoronary blood flow and subsequent myocardial ischemia. Thus, a methodaccording to the present invention to ameliorate the ischemia duringcoronary angioplasty involves infusing an oxygenated microbubbleformulation into the distal coronary artery during balloon inflation.References which demonstrate the usefulness of liquid PFC emulsions inminimizing the ischemia from coronary angioplasty include Jaffe et al.,1988, Am Heart J 115:1156-64; Lowe, 1991, supra; Thoolen et al., 1993,Biomater Artif Cells Immobilization Biotechnol 21:53-62; Cleman et al.,1986, Circulation 74:555-62; Kerins, 1994, Am J Med Sci 307:218-21;Kloner and Hale, 1994, supra; Robalino et al., 1992, J Am Coll Cardiol20:1378-84; and Garrelts, 1990, DICP 24:1105-12. Myocardial reperfusioninjury is ischemic injury following a myocardial infarct (aninterruption of coronary blood flow with subsequent myocardialischemia). Thus, a method according to the present invention toameliorate the ischemia following a myocardial infarct involves combinedtherapy involving oxygen inhalation and intracoronary and intravenousinfusions of the microbubble formulation in the perireperfusion period.Alternatively, the therapy may comprise of infusions of oxygenatedmicrobubble formulation, alone, including selective aortic archperfusion during cardiac arrest. References which demonstrate theusefulness of liquid PFC emulsions in minimizing the ischemia frommyocardial reperfusion injury include Kloner and Hale, 1994, supra;Garretts, 1990, supra; Forman et al., 1992, Am Heart J, 124:1347-57;Manning et al., 1992, Ann Emerg Med 21:1058-65; Martin et al., 1992,Biomater Artif Cells Immobilization Biotechnol 20:985-9; and Forman etal., 1991, J. Am. Coll. Cardiol. 18:911-8.

Solid tumor masses may often contain areas of hypoxia which areresistant to anticancer therapy. Since radiation therapy and manychemotherapeutic agents (collectively referred to as "anticancertherapy") are dependent on oxygen to be maximally cytotoxic, one methodaccording to the present invention is to deliver oxygen to the solidtumor masses, thereby enhancing efficacy of anticancer therapy ascompared to therapy without oxygen delivery. The delivered oxygensensitizes hypoxic cells, with little or no observable effect onwell-oxygenated normal tissues. Thus, in a method to deliver oxygen tosolid tumors in a process of enhancing efficacy of anticancer therapy, acombined therapy of oxygen inhalation and administration (injections orinfusions) of the microbubble formulation is used as an adjuvant to theanticancer therapy. Alternatively, a combination of administration ofthe microbubble formulation and hyperbaric oxygen may increase tumoroxygenation and the efficacy of subsequent anticancer therapy.Stabilized microbubbles persisting for less than one hour in thecirculation may be particularly suited for this method. References whichdemonstrate the usefulness of liquid PFC emulsions in enhancing theanticancer therapy of solid tumors include Teacher, 1994, Artif. CellsBlood Substit. Immobil. Biotechnol. 22:1109-20; Evans et al., 1993, IntJ Radiat Oncol Biol Phys 26:649-52; Rockwell et al., 1992, Int J RadiatBiol 61:833-9; Teacher, 1992, Biomater Artif Cells ImmobilizationBiotechnol 20:875-82; Lowe, 1987, supra; Dowling et al., 1992, BiomaterArtif Cells Immobilization Biotechnol 20:903-5; Rockwell et al., 1992,Biomater Artif Cells Immobilization Biotechnol 20:883-93; Teicher etal., 1989, Cancer Res 49:2693-7; and Rockwell, 1994, Artif. Cells BloodSubstit. Immobil. Biotechnol. 22:1097-108.

EXAMPLE 5 Stabilized Microbubbles as O₂ Carrier in Place of Blood

This example is an illustration, additional to the preceding example, ofthe use of stabilized microbubbles in a method of therapeuticallytreating an individual depleted in hemoglobin by delivering oxygen inadequate amounts and at adequate pressures for sustaining therespiratory metabolism of the treated individual. In this illustration,animals were used to demonstrate the efficacy of oxygen delivery by thestabilized microbubbles. The animals were Wistar rats housed and handledaccording to the Helsinki declaration of animal rights. Rats weredivided into three groups: one group, into which was administered thestabilized microbubbles, had normal hemoglobin and normal blood volume;a second group was anemic (lowered hemoglobin but normal blood volume);and a third group was anemic and treated with stabilized microbubblescapable of carrying oxygen. In the following illustration of theinvention, it is important to consider the following concept. The use ofrats has been accepted and validated as an experimental model for theevaluation of in vivo use of microbubbles because the model has beenshown to be predictive of the effectiveness of these agents in humans(See, e.g. D'Arrigo et al., 1993, Invest. Radiol. 28:218-222; Barbareseet al., 1995, J. Neurooncol. 26:25-34; Simon et al., 1992, Invest.Radiol. 27:29-34; Simon et al., 1990, Xnvest. Radiol. 25:1300-1304).

The rats in each group were anesthetized with intraperitonealpentobarbital, 50 mg/kg. Additional doses of 100 mg each were given whenneeded. As illustrated in FIG. 15, the animals were prepared as follows.The animals were trachostomized with a snug-fitting polypropylenecatheter, with care to avoid enlarging the ventilatory dead space.Through a small skin incision, poly-ethylene catheters were introduced 5cm into the left femoral artery and vein. The catheter in the femoralvein was divided into two branches close to the animal by a Y-connectionso that two infusates could be administered simultaneously. Stabilizedmicrobubbles, prepared as previously described, were administered to therats in the form of an emulsion of liquid droplets of theperfluorocarbon dodecafluoropentane (DDFP), wherein at body temperature,the liquid droplets became gas microbubbles. When the perfluorochemicalemulsion was administered, this arrangement allowed for the stabilizedmicrobubbles to traverse only 2 cm before it was inside the animal, aprecaution against premature bubble formation.

Experimental and physiologic parameters were monitored as follows.Arterial blood pressure (AP) and heart rate (HR) were measured with adisposable pressure transducer and continuously recorded on amultichannel recorder. Arterial blood gases were measured with a probeby perfusing arterial blood over sensors via a shunt between the leftfemoral artery and vein. Central venous pressure (CVP) and variations ofCVP due to respiration (dCVP) were continuously recorded on a recorderwith another pressure transducer attached to the catheter that was inthe superior vena cava. A saline-filled catheter connected to a pressuretransducer was introduced into the esophagus to measure the intrapluralpressure (IPP) and respiratory IPP variations (dIPP) indirectly andcontinuously. Muscle P_(O2) was measured in two ways: a sensor, of thesort that is used for transcutaneous O₂ --CO₂ measurements andcalibrated at a temperature of 37.5° C., was placed on an abdominalmuscle through a 2 cm skin incision, with the skin closed above thesensor. The sensor has an interface with a tissue that has a 2 mmdiameter, and thus integrates P_(O2) in regions that are near and farfrom arterioles. Secondly, platinum needle electrodes were polarizedwith 0.7 volt against a Ag/AgCl reference electrode, wherein theplatinum electrodes were placed in muscle tissues on the thorax andabdomen, and the reference electrode was inserted under the skin on theback of the rat. The needle electrodes record P_(O2) in a very small,circumscribed region within the muscle fibers.

Rats in group 1 (animals having normal hemoglobin and normal bloodvolume) were prepared for the experiment using the aforementionedprocedures. After the animals were stabilized, one branch of the venouscatheter was used for infusion of lactated Ringer's solution and theother branch was used for infusion of the stabilized microbubbles whilethe experimental and physiologic parameters were continuously recorded.The liquid emulsion of DDFP droplets for producing stabilizedmicrobubbles were introduced at rates between 0.008 and 0.03 ml/min in amixture with 0.4 ml/min of lactated Ringer's solution. Three rats ingroup 1 were given bolus i.v. injections of 0.2 ml of the liquid DDFPemulsion, and the temperature in the blood of the aortic arch wascontinuously measured with a thermistor implanted through the rightcarotid artery.

For rats in groups 2 and 3, the hemoglobin content of blood was reducedwhile maintaining normal blood volume by withdrawing a volume of bloodand replacing the same volume withdrawn with an infusion of fluid. Thus,one branch of the catheter in the femoral vein was used for infusing 8%bovine albumin in lactated Ringer's solution and the other branch forinfusing either the stabilized microbubbles or a control solution. Thecontrol solution was either saline or the vehicle in which DDFP dropletsare suspended in the process of formulating the stabilized microbubbles(results with the vehicle and saline were not significantly different sothe results were combined). After the animal preparation and whenphysiological values became stable, a blood/albumin exchange wasstarted. Blood was withdrawn from the catheter in the carotid artery ata rate of 0.4 ml/min, and albumin solution was infused into the lowervena cava through the catheter in the femoral vein, also at a rate of0.4 ml/min. Hemoglobin concentration was determined. When a rat'shemoglobin was reduced to 50% of its initial value, the animal wasrandomly assigned into either the second or third group. While theblood/albumin exchange was continued, group 2 animals were infused withthe control solution at a rate of 0.008-0.016 ml/min. While theblood/albumin exchange was continued, group 3 animals were infused withstabilized microbubbles at a rate of 0.008-0.016 ml/min. The stabilizedmicrobubble preparation was infused either directly (4 rats), or througha filter having a pore size of 1.2 μm pores (4 rats); however, resultsin either case were not substantially different, and therefore theresults were combined. In four of the animals that survived for 2 ormore hours with hemoglobin below 15% of the initial value, withdrawnblood was centrifuged to produce high-hematocrit blood, which was thenre-infused at a rate of 0.2 ml/min i.v. to increase the hemoglobin toapproximately 50% of the individual animal's initial value.

Table 3 illustrates the physiological variables for the animals in group1 having normal hemoglobin, normal blood volume, and being treated withthe stabilized microbubbles. Table 4 illustrates the physiologicalvariables for the animals in group 2 having lowered hemoglobin andwithout stabilized microbubble treatment. Tables 5 and 6 illustrate thephysiological variables for the animals in group 3 having loweredhemoglobin and having treatment with the stabilized microbubbles.Abbreviations used in the tables are as follows. AP =arterial bloodpressure; CVP=central venous pressure; DAP=diastolic arterial pressure;dCVP=increase of the central venous pulse caused by an inspiration;dIPP=magnitude of the fluctuation (difference) of intrapleural pressuredue to an inspiration; Hb =hemoglobin concentration; HR=heart rate inbeats/minute; MAP=mean arterial pressure; n=number of animals; PaO₂=partial pressure of O₂ in arterial blood; PaCO₂ =partial pressure ofCO₂ in arterial blood; PmCO₂ =partial pressure of CO₂ in muscles; PmO₂(p)=partial pressure of O₂ in muscles measured with a platinum needleelectrode; PmO₂ =partial pressure of O₂ in muscles with the sensor;PP=pulse pressure; RF=respiratory frequency in breaths/minute;SAP=systolic arterial pressure.

                  TABLE 3                                                         ______________________________________                                        Effect of microbubble infusion in unbled animals (group 1)                               <---------------70% O.sub.2 --------------->                       <-Air->             =======time after infusion=======>                        Air        70% O.sub.2                                                                            5 min.    15 min. 30 min.                                 ______________________________________                                        SAP   152 ± 2                                                                             171 ± 3                                                                             169 ± 4                                                                            161 ± 5*                                                                           167 ± 2                              mmHg                                                                          MAP 123 ± 2  136 ± 3  132 ± 4* 127 ± 4  134 ± 2                                                       mmHg                                   PP 44 ± 2 48 ± 1 54 ± 4 51 ± 5 49 ± 3                          mmHg                                                                          CVP 2.5 2.7 3.2* 3.3 3.2                                                      mmHg                                                                          dCVP 3.3 3.6 4.4*  4.5*  4.6*                                                 mmHg                                                                          HR 414 ± 4  408 ± 2  397 ± 5* 392 ± 8* 392 ± 3*                RF 92 ± 5 76 ± 3 79 ± 5 81 ± 4  96 ± 8*                        dIPP 4.1 5.2 5.6  5.5 5.2                                                     mmHg                                                                          PaO.sub.2 81 ± 4 405 ± 6   426 ± 10*  419 ± 12* 412 ± 14       mmHg                                                                          PaCO.sub.2 48 ± 1 52 ± 2 51 ± 3 49 ± 3  46 ± 1*                mmHg                                                                          PmO.sub.2 56 ± 6 80 ± 4  90 ± 8*  87 ± 11  86 ± 12                                                    (p)                                    mmHg                                                                          PmO.sub.2 56 ± 6 127 ± 7  139 ± 6  186 ± 9* 250 ± 15*                                                 mmHg                                   PmCO.sub.2 50 ± 1 55 ± 3 56 ± 5 54 ± 3  48 ± 1*                mmHg                                                                        ______________________________________                                         *P < 0.05 compared with breathing 70% O.sub.2 before microbubbles infusio

                  TABLE 4                                                         ______________________________________                                        Effect of plasma expander infusion in anemic animals (group 1)                       <-Air->  <--------------70% O.sub.2 -------------->                    <---------------n = 8--------------->                                                                     <-n = 2->                                         100% Hb.sup.+                                                                             100% Hb  75% Hb    25% Hb 15% Hb                                  ______________________________________                                        SAP    155 ± 8                                                                             171 ± 8*                                                                            157 ± 9                                                                            122 ± 10*                                                                         85 ± 3*                              mmHg                                                                          MAP 125 ± 8  138 ± 8  119 ± 11 78 ± 7* 55 ± 3*                 mmHg                                                                          PP mmHg 45 ± 2 50 ± 1  57 ± 3* 66 ± 8* 45 ± 4                  CVP 2.3 2.3 3.1  4.6* 6.5*                                                    mmHg                                                                          dCVP 3.9 4.6 4.5 5.2 6.4*                                                     mmHg                                                                          HR 396 ± 18 386 ± 8  381 ± 20 368 ± 18  419 ± 36                                                      RF 78 ± 7  63 ± 6* 65 ±                                             7 82 ± 12 89 ± 11                 dIPP 4.4 5.4 6.0 5.7 7.4*                                                     mmHg                                                                          PmO.sub.2 72 ± 7 160 ± 18*  140 ± 17* 69 ± 12 13 ± 5*                                                 mmHg                                   PmCO.sub.2 49 ± 4  58 ± 3*  69 ± 6*  69 ± 10* 62 ± 7*                                                 mmHg                                 ______________________________________                                         *P < 0.05 compared to breathing of air                                        .sup.+ 100% Hb for this group 17.2 ± 0.5 g/100 ml                     

                  TABLE 5                                                         ______________________________________                                        Anemic Animals During Microbubble Treatment                                              <---------------70% O.sub.2 --------------->                       <-Air->              =====microbubble infusion=====>                          100% Hb.sup.+                                                                            100% Hb   50% Hb    25% Hb 15% Hb                                  ______________________________________                                        SAP   159 ± 8                                                                             181 ± 6*                                                                             169 ± 3                                                                            144 ± 2*                                                                          115 ± 5*                             mmHg                                                                          MAP 127 ± 7 146 ± 3* 125 ± 3 99 ± 2* 76 ± 2*                   mmHg                                                                          PP 48 ± 4 51 ± 4  66 ± 2* 68 ± 3* 58 ± 4                       mmHg                                                                          CVP 3.3 3.4 4.1 4.6 5.6*                                                      mmHg                                                                          dCVP 3.9 4.8 4.6 4.6 4.8*                                                     mmHg                                                                          HR  397 ± 14 417 ± 15 431 ± 20 421 ± 17  397 ± 17                                                     RF 66 ± 3 58 ± 5 64 ± 5                                             71 ± 6  72 ± 6                    dIPP 4.7 5.2 6.2* 6.0 6.2*                                                    mmHg                                                                          PmO.sub.2 73 ± 6  174 ± 22*  125 ± 22* 97 ± 14 70 ± 10                                                mmHg                                   PmCO.sub.2 54 ± 2  65 ± 4*  68 ± 9* 66 ± 4* 69 ± 5*                                                   mmHg                                 ______________________________________                                         *P < 0.05 compared to breathing of air                                        .sup.+ 100% Hb for this group 18.0 ± 0.7 g/100 ml                     

                  TABLE 6                                                         ______________________________________                                        Anemic Animals (from Table 5) After Microbubble Treatment; with                 two hour follow up at low Hemoglobin                                                <---Time at 13% of control Hb, breathing 70% O2--->                               10 min.  40 min.                                                                              80 min.                                                                              100 min.                                                                             120 min.                            ______________________________________                                        SAP mmHg                                                                              112 ± 5*                                                                            123 ± 4*                                                                            120 ± 4*                                                                          118 ± 5*                                                                          118 ± 4*                             MAP mmHg 73 ± 3* 78 ± 2* 77 ± 3* 73 ± 4* 72 ± 3*                                                      PP mmHg 59 ± 4* 67 ± 4* 65                                             ± 4* 68 ± 5* 69 ± 4*                                                  CVP mmHg 5.6* 4.3 3.7 3.5 3.3                                                 dCVP 4.9  4.8 4.0 4.2 5.1                                                     mmHg                                   HR 394 ± 16  407 ± 18  425 ± 18  427 ± 17  441 ± 12*                                                  RF 73 ± 6  80 ± 8* 83 ±                                             7* 81 ± 7* 86 ± 4*                dIPP mmHg 6.6*  6.4* 5.8 6.1 6.1                                              PmO.sub.2 mmHg 54 ± 8  48 ± 8  45 ± 7  42 ± 8  29 ± 6*                                                PmCO.sub.2 67 ± 6* 62 ± 6                                              59 ± 5  58 ± 8  48 ± 2                                                mmHg                                 ______________________________________                                         *P < 0.05 compared to breathing of air                                   

Physiologic parameters for animals breathing air before any infusionappears in the first column of data in Tables 3-5, wherein such valuescorrespond well with expected values reported for anesthetized animalsbreathing air. The second data column of Tables 3-5 shows that theresponse to increased O₂ in the breathing gas was similar amongst thegroups of animals. Arterial PaO₂ and PaCO₂, SAP, MAP, and muscle P_(O2)rose, and respiratory frequency (RF) fell, reaching maximal responses in2 to 5 minutes. Table 3 shows that PaO₂ and PaCO₂ increased to 405 and52 mmHg, respectively; both these values are significantly differentfrom control with p value less than 0.01.

As demonstrated by the responses illustrated in Table 3, theadministration of stabilized microbubbles to rats having normalhemoglobin demonstrated that the microbubbles could increase thearterial P_(O2), and that the microbubbles lasted in the body for anhour or more. For example, five minutes after intravenous infusion ofstabilized microbubbles was started, average arterial PaO₂ was increasedby 5%- (Table 3). The muscle PmO₂ measured with the probes increasedmarkedly, and PmO₂ measured with platinum electrodes increased at 12 of16 sites by an average of 10 mmHg. Arterial PO₂ tended to level at 420mmHg and P_(CO2) decreased from 55 to 48 mmHg. In 3 experiments in whichmeasurements were made in the vena cava with a platinum needleelectrode, the venous O₂ remained at the control value throughout theentire experiment. At the end of the infusion, 0.5 to 0.8 ml ofstabilized microbubbles had been given. The tissue PO₂ remained high forat least 120 minutes after the infusion was stopped, appearing torepresent microbubble gas delivery function due to stabilizedmicrobubbles remaining in the circulation until eventual clearance fromthe body via the lungs. FIG. 16 illustrates the time course in oneanimal of the elevation of abdominal muscle P_(O2). P_(O2) (O) beganelevating during the 20 minute infusion of stabilized microbubbles (₋₋)and remained elevated for more than 2 hours.

After infusion of approximately 0.3-0.6 ml at 50 μl/min of the DDFPdroplet emulsion for producing stabilized microbubbles, the AP fell, CVPincreased, and the animals changed their breathing pattern from auniform frequency and depth to a pattern having an occasional very deepgasping breath along with normal breaths. As the microbubble infusioncontinued, the irregular breathing pattern became more obvious, thegasps became more frequent, AP fell, and the CVP increased. Thebreathing irregularity increased in frequency for 30 minutes aftermicrobubble infusion ceased, after which the breathing pattern graduallynormalized over the next 2 hours. The irregular breathing wastentatively ascribed to pulmonary gas embolism caused by coalescence ofmicrobubbles. After the 2 hours, while the breathing gas was 70%, O₂,the arterial O₂ fell gradually to the starting level. When the rats wereallowed to breathe air, the arterial O₂ fell to the same level as thelevel when breathing air at the start of the experiment. The tissueelectrode readings fell to lower values than in the beginning of theexperiments. In air, the RF increased above the pre-exposure control,and was associated with a marked hyperventilation, as indicated by thefall of PaCO₂ to 11-12 mmhg.

In summary, in animals having normal hemoglobin levels, infusion ofstabilized microbubbles resulted, within minutes, in increased arterialand muscle O₂ partial pressures. Rhythmic fluctuations in muscle O₂partial pressures were observed, possibly due to vasomotion. The higharterial P_(O2) may cause autoregulatory blood flow reduction in thetissue, which tends to keep the venous O₂ unchanged.

As demonstrated by the responses illustrated in Table 4, withoutadministration of stabilized microbubbles to rats having loweredhemoglobin but normal blood volume (via albumin infusion), the conditionof the animals worsened significantly as Hb decreased; SAP decreasedmarkedly, P_(O2) in the muscles fell toward zero, and P_(CO2) in themuscle rose. With vehicle/albumin infusion to replace blood volume, thecondition of the animals deteriorated completely when hemoglobin was 20%to 15% of the initial value. In contrast, as demonstrated by theresponses illustrated in Table 5, rats that had the same reduction ofblood hemoglobin but were given stabilized microbubbles in addition tothe albumin infusion, showed physiological measures that remained normalduring the infusion of stabilized microbubbles, and also after infusionwas stopped. Muscle P_(O2) and PCO_(CO2) remained normal or above normaluntil the end of the exchange. PP increased by the same magnitude as itdid in the group given vehicle/saline, but the SAP, MAP, and CVP werefairly constant (Tables 5 and 6). By the time the infusion of the DDFPemulsion was stopped, there were deep breaths at a rate of approximately1 to 3 in 5 minutes. The deep breaths increased in frequency for 15minutes after microbubble infusion ceased, and then gradually normalizedover the next 2 hours. In several later series of studies on ratsaccording to essentially the same protocol the infusion rate of the DDFPemulsion was reduced to 10 μl/min. No respiratory irregularitiesoccurred in these rats. This was thought to be the result of themicrobubbles being more dispersed and less likely to coalesce and formpulmonary emboli. The total amount of liquid emulsion of DDFP dropletsgiven to produce stabilized microbubbles was 0.08-0.11 ml/100 g of ratweight. All the animals receiving microbubbles had stable conditions for2 or more hours even though their hemoglobin was only 2.0-2.4 g/100 ml(13% of the initial values).

Four of the animals having been infused with stabilized microbubbleswere re-transfused with blood with high hematocrit after the anemiaepisode. As a result, PP fell toward normal and AP, muscle O₂, andmuscle CO₂ increased during the re-transfusion. The re-transfused ratswere given 70% O₂ to breathe for 1 hour at 50-60 of initial Hb values.After 1 hour in 70% O₂, they were exposed to air for 1 hour; wherein AP,PP, CVP, dIPP, and muscle gas partial pressures remained constant at thesame levels as during air breathing before the experiments. Thus, theseanimals lived for 2 hours after the re-transfusion, and they had shownno signs of deterioration for the time period in which their conditionswere followed.

In summary, all of the rats with infusions of stabilized microbubbles,having the same low Hb and also breathing 70% O₂, obtained adequate O₂via the microbubbles so that they survived for more than two hours.Tissue O₂ partial pressures stayed at or near the level found in thecontrol situation when the animal had breathed air. There was noincrease in arterial or tissue CO₂ partial pressures over that due tobreathing of 70% O₂. Taken together with measured physiologic values ofthe animals of group 1, the results indicate that the microbubbles havea long physiological half life of more than 2 hours.

Precautions should be taken when administering stabilized microbubbles.For example, for DDFP, warming the solution to body temperature beforeinjection could result in the appearance of large bubbles which maycause undesirable effects when administered. Likewise, animals tendednot to tolerate well administration of DDFP suspensions that hadpreviously been subjected to temperatures characteristic ofrefrigeration. Additionally, the rate of DDFP emulsion or stabilizedmicrobubble infusion should be appropriately slow to avoid possibleincrease in size of the bubbles by coalescence to diameters that maycause embolization of blood vessels either in the lungs or in thetissues.

EXAMPLE 6 Method of Using Stabilized Microbubbles as a Carrier ofAnesthetic Gas

Stabilized microbubbles, such as those formulated from slowly permeatinggas, may be used to carry anesthetic gases to and from tissues, with theintent of delivering such gases in a rapid and controlled manner, in amethod of anesthetizing an individual rapidly and in reversing theanesthetized state rapidly. Generally, anesthetic gases are more solublein blood than N₂. According to this method of the present invention, atherapeutically-effective amount of microbubbles is introduced into theblood circulation of an individual, wherein the microbubbles function tocarry anesthetic gas(es) from the lungs in adequate amounts and atadequate pressures to allow exchange with tissues to anesthetize thetreated individual. One skilled in the art would appreciate that theamount of microbubbles to be introduced (number of microbubbles/ml)depends on a number of factors including the particular stabilizingmechanism used for microbubble preparation, the size (radius) range ofthe microbubbles in the preparation, the volume of blood in theindividual to be treated, and the efficacy of the anesthetic gas carriedby the microbubbles. For example, if a microbubble carries 60% N₂ O, andif N₂ O solubility is 0.45 ml N₂ O ml blood/atm and blood PN₂₀ is 0.8atm, then the microbubble would carry approximately 1.5 times more N₂ Othan an equal volume of blood. The maximal enhancement of solubility forN₂ O would be 1/0.45=2.2. Potentially such solubility enhancement willbe magnified by differences in diffusional transport from blood totissues mediated by the presence of microbubbles. The therapeuticallyeffective amount of microbubbles, containing one or more anestheticgases, can be administered into the blood circulation of the individualto be treated by methods known in the art, including intravenousadministration.

EXAMPLE 7 Method of Using Stabilized Microbubbles to Remove Inert GasFrom Tissues

Stabilized microbubbles, such as those formulated from slowly permeatinggas, may be used to carry nitrogen or other inert gas in a breathingmixture out of tissue, in a method of preventing or absorbing dangerousbubbles which contain inert gas. Such bubbles are encountered in gaseousemboli, and are the initial cause of decompression sickness inunderwater divers, aviators in unpressurized aircraft, and astronautsengaged in extravehicular activities. Carriage of inert gas, such as N₂,from tissue to the lung by microbubbles in the blood is a logicalcorollary of the idea that microbubbles can carry oxygen. Referencesthat demonstrate the usefulness of liquid PFC emulsions indenitrogenating tissue are Cassuto et al., 1974, Aerospace Med 45:12-14;Novotny et al., 1993, J Appl Physiol 74:1356-1360; and Speiss et al.,1988, Undersea Biomed Res 15:31-37. Microbubbles can be expected to bemore efficacious and more practical than perfluorocarbons.

FIG. 17 illustrates that when N₂ is present in tissue of a person whobreathes pure O₂, the microbubble will accumulate large amounts of N₂ asit passes through the tissue and will unload N₂ in the lungs. The amountof N₂ carried is about two-thirds of the amount of O₂ carried. Themicrobubbles can carry much more N₂ than blood. If N₂ comprises 60% of amicrobubble in venous blood of a person breathing pure O₂, as in FIG.17, the microbubble will carry 0.6 ml N₂ /ml gas/atm. Contrasted withblood solubility of N₂ of 0.015 ml N₂ /ml blood/atm and assuming thatP_(N2) of blood is 0.8 atm, it is seen that the microbubble can carry 50times more N₂ than the same volume of blood.

EXAMPLE 8 Use of Stabilized Microbubbles in Right-to-Left Shunts

This embodiment illustrates the use of the microbubbles of the presentinvention in alleviating the effects of a right-to-left circulatoryshunt (RLS). To illustrate this embodiment, RLS was created in pigs,which is an accepted animal model for such studies because their lungslack interlobular vascular connections. Pigs weighing between 32-160pounds were anesthetized by standard procedures. The trachea wasexposed, opened and a cuffed tracheal tube was introduced. The rightfemoral artery and vein were cannulated and arterial pressure (AP) andheart rate (HR) were measured continuously using a COBE disposablepressure transducer (COBE, Lakeweood, Colo.). A constant, continuousintravenous infusion of 1 ml/min of lactated Ringer's solution andsustaining doses of sodium pentobarbital were given. Samples wereobtained from the left femoral artery for arterial blood gasmeasurements. Blood gases were measured at 5-15 min intervals usingstandard measuring equipment (Ciba-Corning Blood Gas System, model 278).Stabilized bubbles in lactated Ringer's solution was infused via theleft femoral vein. The right external jugular vein was exposed andcannulated with a Swan-Ganz catheter (size 5 F). The catheter wasintroduced into the right cardiac ventricle, the balloon partly inflatedand floated in the blood stream until positioned in the pulmonaryartery. The pulmonary arterial pressure (PAP) was measured using a COBEdisposable pressure transducer. In some cases the Swan-Ganz catheter tipwas placed in the right ventricle to measure the right ventricularpressure (RVP). A catheter was placed in the right external jugular veinalongside the Swan-Ganz catheter with the tip in the right atrium tomeasure the central venous pressure (CVP) and the respiratory frequency(RF). The left external jugular vein was cannulated and the catheterintroduced into the superior vena cava. Blood samples were obtained fordetermination of venous blood gases. Two transcutaneous O2-CO2combi-sensors (Kontrol, Zurich, Switzerland, and Radiometer, Copenhagen,Denmark) were placed in small pouches over abdominal muscle tissue forcontinuous measurements of tissue O2 and CO2.

Following surgery, the pigs were allowed to stabilize for a period of 30min while breathing air. Measurements of AP, PAP, CVP, RF, and HR wererecorded over the next 15 min. At least two sets of control bloodsamples were collected for arterial and venous blood gas analysis. Theblood was sampled with airtight glass syringes and analyzed within 20seconds. The circulatory RLS was established as follows.

In Group I, a Swan-Ganz catheter was introduced into one main bronchus,and the cuff inflated in order to close off that part of the lung. Thisreduced the arterial O₂ tension (PaO2) somewhat, but inhalation of ahigh concentration of oxygen increased the PaO2 regularly to above 250mmHg.

In Group II, in 8 pigs, steel or glass beads of about 1 mm diameter wereinjected into the bronchial tree, creating atelectasis by multiple endobronchial obstructions (Eyal et al., 1996), until the arterial PaO2 hadfallen to approximately to 30 mm Hg. This procedure caused a morepronounced RLS since inhalation of pure O2 could only partly compensatefor the applied shunt, limiting the rise in the PaO2 to less than 70 mmHg.

While the pigs breathed air, after the shunt was established, arterialand central venous blood samples were taken, analyzed and shunt fractioncalculated according to standard methods (Levitzky 1991). At the end ofthe air breathing period, the animals were given pure oxygen to breathe.During the first 20-30 min of O₂ breathing, new baseline values wereestablished and then the I.V. infusion of stabilized microbubbles wasstarted at a rate of 0.1-0.2 ml/min in 4 ml/min lactated Ringer'ssolution. A total of 2-6 ml of microbubbles was given during the next 30min. Arterial blood samples were taken and analyzed for blood gasesevery 5 min during the infusion period and every 10 min for the rest ofthe experimental period. The criterion for terminating the stabilizedmicrobubbles infusion was a substantial increase in arterial PaO₂ andtissue PO₂ tension. Regularly, the maximal effect was seen 50-80 minafter the infusion of DDFP emulsion was terminated and started to wearoff 150-300 min later. The blood gases gradually returned to thebaseline value. In seven pigs, a second and third dose of 2-5 ml wereinfused in an identical manner as described above until a rise in PaO₂was established. Blood samples were analyzed for the next 5-12 threehours.

The spontaneously breathing pigs were administered 100% O₂ before,during, and after stabilized microbubbles infusion. A schematic overviewof the experiments is indicated by the time lines in the figures andtables.

At the end of the experiments, the pigs were sacrificed with a lethalI.V. dose of pentobarbital sodium (100 mg/kg). No adverse effects ofstabilized microbubbles infusion were found on HR, RF, and bloodpressure during any of the experiments (maximal duration 16 h in sevenpigs). The body temperature was kept within 1° C. of the coretemperature observed at the start of each specific experiment.

During air breathing control periods, all blood pressures, HR, and RFwere similar and within normal ranges (Tables 7 and 8) in the two typesof shunts. Though the PaO₂ was somewhat different in the two groups,i.e. 66.2±3.7 (mean+SE) mm Hg in Group 1 and 80.3±3.0 mm Hg in group 2,the O₂ saturation was similar and normal, at 93.8+0.3 and 95.3+0.5%,respectively (Tables 9 and 10, FIG. 19). At the same time, PaCO₂ was45.1±4.0 and 41.8±1.3 mm Hg in the two groups (Tables 10 and 11, FIG.22. The muscle PO₂ was 58±8 (Kontron electrode) and 59±3 mm Hg(Radiometer electrode) in Group 1 and 44±5 and 41±8 mm Hg in Group 2(Tables 9 and 10, FIGS. 20, and 21), and the tissue PCO₂ was 66±3 and62±2, and 69±2 and 55±3 mm Hg, respectively (Tables 9 and 10, FIGS. 23and 24).

The shunt fractions were calculated to be 0.27 in one representativeanimal in Group 1 and 0.20±0.02 (SE) in Group 2.

Establishment of Pulmonary Shunt

When one lung or parts of one lung was closed off from the ventilationby an inflated balloon in Group 1 pigs, PaO₂ fell to 45.7±1.5 mm Hgreducing the O₂ saturation to 80.8±3.4%, and local muscle PO₂ to 40±2and 37±5 mm Hg. The PaCO₂ increased to 51.1±5.4 mm Hg, and tissue PCO₂was raised to 66±4 and 61 ±5 mm Hg. The shunt fraction increased to0.43. When beads were used to block ventilation (Group 2 pigs), PaO₂fell to 32.2 ±2.2 mm Hg and arterial O₂ saturation to 61.2±5.9%. Themuscular PO₂ fell to 23±5 and 20±5 mm Hg. The PaCO₂ increased to63.1±2.5 mm Hg and muscular PCO₂ to 98±9 and 81±10 mm Hg. The shuntfraction increased to 0.57±0.06 (SE).

When O₂ breathing was established, the PaO₂ increased in both groups ofanimals, but to different levels. The PaO₂ in Group 1 animals increasedto 216.6±15.2 mm Hg (FIG. 19) and an oxygen saturation of 99.5±0.1% wasobtained, whereas in Group 2 animals PaO₂ was also increased, but didnot reach the control level in air (68.6±9.1 mm Hg) (FIG. 19) giving asaturation of 89.5±5.3%.

During the initial phase of O₂ breathing, the respiration ceased in the3 animals which had the most marked increases in PaCO₂ and artificialventilation had to be administered for the next 5-15 min.

Infusion Id Microbubbles Aqenerating DDFP Emulsion

During infusion of DDFP emulsion, the PaO₂ started to increase within 1min of infusion or 0.1 ml, and continued to rise during the infusion inboth animal groups (FIG. 19). The 3 animals with high PaCO₂ andrespiratory arrest, started to breathe spontaneously again after 5 minof infusion of DDFP. In all animals in Group 2, PaCO₂ and tissue PCO₂fell during the infusion (FIG. 22).

After infusion of DDFP emulsion less than 1 ml (10 min of infusion), thePaO₂ was significantly increased wether the initial PaO₂ was hyperoxic(>150 mm Hg of O₂) or hypoxic (<80 mm Hg of O₂) . At the same time themuscle PCO₂ fell on the two monitoring devices (FIGS. 21-24).

The PaO₂ increased steadily over the next 20 min in both groupswhereupon it remained at between 130 and 350% of the initial value forthe remaining infusion period and for the next 2 h (FIG. 19). After aperiod of 2-3 h, the PaO₂ and muscle P0₂ (FIGS. 20,21) began to declineand PaCO₂ (FIG. 22) and muscle PCO₂ (FIGS. 23,24) increased. Dependingon the amount of infused DDFP emulsion, the control levels of O₂ and CO₂were approached after approximately 3 h.

The systolic AP, mean AP and diastolic AP remained stable and within 10mm Hg of the control values during the experiments (Tables 7,8). The HRremained constant throughout the experiments. The changes provoked byinducing the RLS, were all corrected during the infusion of DDFPemulsion so that RVP, CVP, and PAP remained mainly unaltered throughoutthe experimental period (Tables 7,8).

FIG. 25 shows the general effect of an EchoGen® infusion on PaO₂ whenall infusions in Group 2 pigs were summarized, whether they were thefirst, 2nd, or 3rd. This graph indicates an effective O₂ carryingcapacity of the DDFP micro-bubbles exceeding 4 h.

When repeated doses of microbubbles were given in seven of Group 2animals as shown in FIG. 26, the PaO₂ followed the same pattern asduring and after the first infusion, though higher levels were obtainedwith a lower dose of infused DDFP emulsion. Furthermore, the high PaO₂tended to be sustained longer after the 2nd and 3rd infusion than afterthe first infusion.

These results demonstrate that stabilized microbubbles in combinationwith oxygen breathing, is capable of effectively counteracting theadverse effects of severe right-to-left circulatory shunt on gasexchange. With the doses used, animals that before treatment wereseverely hypoxic, hypercapnic, and hypertensive, became hyperoxic,nearly normocapnic and their circulatory parameters normalized tocontrol levels. Already one minute after instituting the therapy, thePaO₂ began to increase and after 10 min it had reached 155% (106.5±15.1mm Hg, p<0.01) of the control level (68.2±9.1 mm Hg) and within 150 minit had reached approximately 200%.(134.9±9.1 mm Hg, p<0.01).

Those skilled in the art will recognize that a considerably lower rateof infusion can be used while still attaining adequate O₂ and CO₂exchange. It should be noted that even with the intense treatment usedin this study, no adverse effects of microbubbles infusion were observedalthough they were repeated up to 3 times and the animals were monitoredup to 12 hours.

The absence of side effects in this large animal model and the longduration (4h) of the treatment effect indicates that this application ofstabilized microbubbles can be used in the treatment of right to leftshunt as described herein.

                                      TABLE 7                                     __________________________________________________________________________    Blood pressures, heart rate and respiratory frequency in animals treated      with EchoGen ® for                                                          right-to left circulatory shunts in the lungs induced by partial airway     blockage with an                                                               airfilled balloon. The shunt fraction increased from 0.27 to 0.43 after      the balloon was                                                                 inflated (measured in one representative animal).                                                HR   RF  PAP PAP PAP RVP         CVP                       SAP DAP MAP beats/ breaths/ sys dia mean max RVP ed dCVP mean                 mm Hg mm Hg mm Hg min min mm Hg mm Hg mm Hg mm Hg mm Hg mm Hg mm            __________________________________________________________________________                                                          Hg                      Control                                                                             135 ± 5                                                                         88 ± 6                                                                          104 ± 6                                                                          148 ± 14                                                                       24 ± 2                                                                         28 ± 2                                                                         21 ± 2                                                                         22 ± 2                                                                         25  0   5 ± 1                                                                          4 ± 2                  Shunt                                                                         Induced                                                                       air  162 ± 91  10 ± 11 127 ± 8 178 ± 9 26 ± 3 28 ± 1                                                            22 ± 1 24 ± 1                                                           40 1 5 ± 1 4                                                               ± 1                    breathing                                                                     oxygen 135 ± 6 90 ± 4 105 ± 4 156 ± 8 26 ± 3 27 ± 1                                                             22 ± 1 24 ± 1                                                           26 -2  4 ± 0 3                                                             ± 1                    breathing                                                                     EchoGen ®                                                                 Infusion                                                                      10 min 125 ± 5  75 ± 4*  92 ± 4  163 ± 11 24 ± 3 28 ±                                                           2 22 ± 1 24 ±                                                           1 22 3 3 ± 0 6                                                             ± 3                    at end of 137 ± 3 87 ± 5 103 ± 4 183 ± 4 26 ± 4 26 ±                                                            1 18 ± 1 20 ±                                                           1 26 2 2 ± 0 4                                                             ± 2                    infusion                                                                      After 140 ± 4 90 ± 5 107 ± 5  178 ± 10 24 ± 4 27 ± 1                                                            18 ± 1 21 ± 1                                                           25 2 3 ± 0 5                                                               ± 2                    Infusion                                                                      30 min                                                                      __________________________________________________________________________     All values are means ± SE, n = 4, *p < 0.05, baseline values during        oxygen breathing versus values during and after EchoGen ® infusion.       SAP -- systolic arterial pressure; DAP -- diastolic arterial pressure; MA     -- mean arterial pressure; HR -- heart rate; RF -- respiratory frequency;     PAPsys -- systolic pulmonary arterial pressure; PAPdia -- diastolic           pulmonary arterial pressure; PAPmean -- #mean pulmonary arterial pressure     RVPmax -- maximal right ventricular pressure; RVPed -- end diastolic righ     ventricular pressure; dCVP -- central venous pulse pressure; CVPmean --       mean central venous pressure.                                            

                                      TABLE 8                                     __________________________________________________________________________    Blood pressures, heart rate and respiratory frequency in animals treated      with EchoGen ® for                                                          severe right-to left circulatory shunts in the lungs induced by             intratrachael injections of                                                     steel beads. The shunt fraction increased from 0.20 ± to 0.02 in         control to 0.57 ± 0.06                                                       after beads were given.                                                                          HR   RF  PAP PAP PAP RVP         CVP                       SAP DAP MAP beats/ breaths/ sys dia mean max RVP ed dCVP mean                 mm Hg mm Hg mm Hg min min mm Hg mm Hg mm Hg mm Hg mm Hg mm Hg mm            __________________________________________________________________________                                                          Hg                      Control                                                                             137 ± 8                                                                          93 ± 5                                                                         108 ± 6                                                                         165 ± 10                                                                        40 ± 5                                                                         17 ± 2                                                                         9 ± 4                                                                          12 ± 3                                                                         32 ± 4                                                                         4 ± 1                                                                          4 ± 1                                                                          4 ± 1                  Shunt                                                                         Induced                                                                       air breathing 163 ± 8 103 ± 8 123 ± 7 179 ± 9  28 ± 4 19                                                           ± 3 8 ± 2 11                                                            ± 2 43 ± 5 8                                                            ± 2 6 ± 1 5                                                             ± 1                    O.sub.2 breathing 147 ± 7  98 ± 5 114 ± 5 176 ± 7  34 ±                                                            6 15 ± 3 7 ±                                                            2 10 ± 3 33 ±                                                           3 6 ± 1 6 ± 1                                                           3 ± 1                  EchoGen ®                                                                 Infusion                                                                      10 min 141 ± 6  96 ± 4 111 ± 4 181 ± 9  36 ± 3 18 ± 3                                                           8 ± 2 11 ± 2                                                            32 ± 3 4 ± 1                                                            5 ± 1 3 ± 0                                                              at end of 145 ±                                                           5  99 ± 4 115                                                              ± 4 185 ± 10                                                            39 ± 4 19 ± 2                                                           9 ± 2 12 ± 2                                                            33 ± 3 4 ± 1                                                            5 ± 1 2 ± 0                                                              infusion                 After                                                                         Infusion                                                                      30 min 149 ± 6 100 ± 5 116 ± 5 186 ± 10 38 ± 4 18 ± 3                                                           9 ± 2 12 ± 2                                                            33 ± 3  3 ±                                                             0* 7 ± 2 3 ±                                                            1                         60 min 155 ± 6 107 ± 4 123 ± 4 182 ± 12 39 ± 4    35                                                               ± 3 4 ± 1 7                                                             ± 1 2 ± 1                                                                90 min 153 ± 7                                                            111 ± 6 125 ±                                                           8 186 ± 10 34                                                              ± 5    35 ± 2                                                           4 ± 1 6 ± 1                                                             2 ± 1*                 120 min ±9 108 ± 6 121 ± 7 178 ± 8  34 ± 6    35 ± 4                                                            4 ± 1 7 ± 1                                                             2 ± 1*                 150 min  151 ± 11 106 ± 7 121 ± 8 175 ± 11 28 ± 7    34                                                            ± 3 4 ± 1 6                                                             ± 1  2 ±          __________________________________________________________________________                                                          1*                       All values are means ± SE, n = 4, *p < 0.05, baseline values during        oxygen breathing versus values during and after EchoGen ® infusion.       SAP -- systolic arterial pressure; DAP -- diastolic arterial pressure; MA     -- mean arterial pressure; HR -- heart rate; RF -- respiratory frequency;     PAPsys -- systolic pulmonary arterial pressure; PAPdia -- diastolic           pulmonary arterial pressure; PAPmean -- #mean pulmonary arterial pressure     RVPmax -- maximal right ventricular pressure; RVPed -- end diastolic righ     ventricular pressure; dCVP -- central venous pulse pressure; CVPmean --       mean central venous pressure.                                            

                                      TABLE 9                                     __________________________________________________________________________    Arterial blood gases, acid base chemistry and tissue O.sub.2 and CO.sub.2     tensions in animals treated                                                    with EchoGen ® for severe right-to-left circulatory shunts in the        lungs induced by partial                                                        airway blockage with an airfilled balloon. The shunt fraction increased     from 0.27 in control                                                           to 0.43 after balloon was inflated (measured in one representative           animal).                                                                             PaO.sub.2                                                                            PaCO.sub.2 HCO.sub.3                                                                          O.sub.2 sat                                                                         PO.sub.2 K                                                                         PO.sub.2 R                                                                         PCO.sub.2 K                                                                       PCO.sub.2 R                   mm Hg mmHG pH mm/l % mm Hg mm Hg mm Hg mm Hg                                __________________________________________________________________________    Control                                                                              62.2 ± 7                                                                          45.1 ± 4.0                                                                      7.45 ± 0.06                                                                      27.5 ± 2.5                                                                      93.8 ± 0.3                                                                       58 ± 8                                                                          59 ± 3                                                                          66 ± 3                                                                         62 ± 2                     Shunt Induced                                                                 air breathing 45.7 ± 1.5  51.1 ± 5.4 7.41 ± 0.07 28.4 ± 2.0                                                       80.3 ± 3.4 40 ± 2                                                       37 ± 5 66 ± 4 61                                                        ± 5                        O.sub.2 breathing 216.6 ± 15.2  53.2 ± 2.1 7.37 ± 0.09 31.7                                                          ± 1.3 99.5 ± 0.1                                                        64 ± 8  80 ± 12                                                         80 ± 5 71 ± 8                                                            EchoGen ®                Infusion                                                                      10 min 332.5 ± 23.8** 53.5 ± 2.4 7.44 ± 0.06 32.3 ± 1.3                                                           99.7 ± 0.1 70 ± 6                                                        86 ± 10 77 ± 9                                                         74 ± 9                     at end of 344.2 ± 29.5** 53.7 ± 1.9 7.45 ± 0.04 31.9 ± 1.4                                                        99.8 ± 0.1 77 ± 7                                                       80 ± 7 66 ± 8 66                                                        ± 9                        infusion                                                                      After Infusion 309.6 ± 32.5** 53.0 ± 2.0 7.42 ± 0.04 32.1 ±                                                       1.4 99.7 ± 0.1 76                                                          ± 8 63 ± 6 68                                                           ± 9 65 ± 9                                                               30 min                     __________________________________________________________________________     All values are means ± SE, n = 4, *p < 0.05, baseline values during        oxygen breathing versus values during and after EchoGen ® infusion.       PaO.sub.2 -- oxygen tension in arterial blood; PaCO.sub.2 -- carbon           dioxide tension in arterial blood, pH and [HCO.sub.3 ] measured in            arterial blood; O.sub.2 sat -- oxygen saturation in arterial blood;           PO.sub.2 K and PO.sub.2 R -- oxygen tension in abdominal  #muscle measure     with Kontron's and Radiometer's transcutaneous combisensors, respectively     PCO.sub.2 K and PCO.sub.2 R -- carbon dioxide tension in abdominal muscle     measured with Kontron's and Radiometer's transcutaneous combisensors,         respectively.                                                            

                                      TABLE 10                                    __________________________________________________________________________    Arterial blood gases, acid base chemistry and tissue O.sub.2 and CO.sub.2     tensions in animals treated                                                    with EchoGen ® for severe right-to-left circulatory shunts in the        lungs induced by intra                                                          tracheal injections of steel beads. The shunt fraction increased from       0.27 in control to                                                              0.43 after balloon was inflated (measured in one representative             animal).                                                                             PaO.sub.2                                                                            PaCO.sub.2 HCO.sub.3                                                                          O.sub.2 sat                                                                         PO.sub.2 K                                                                         PO.sub.2 R                                                                         PCO.sub.2 K                                                                       PCO.sub.2 R                   mm Hg mmHG pH mm/l % mm Hg mm Hg mm Hg mm Hg                                __________________________________________________________________________    Control                                                                              80.3 ± 3.0                                                                        41.8 ± 1.3                                                                      7.44 ± 0.01                                                                      28.3 ± 0.9                                                                      95.3 ± 0.5                                                                       44 ± 5                                                                          41 ± 8                                                                          69 ± 2                                                                         55 ± 3                     Shunt Induced                                                                 air breathing 32.2 ± 2.2  63.1 ± 2.5 7.29 ± 0.02 30.4 ± 1.2                                                       61.2 ± 5.9  23 ±                                                        5  20 ± 5 98 ± 9                                                        81 ± 10                    O.sub.2 breathing 58.6 ± 9.1  50.8 ± 2.6 7.34 ± 0.04 27.9 ±                                                       1.4 89.5 ± 5.3  60                                                         ± 10 49 ± 6 83                                                          ± 5 78 ± 8                                                               EchoGen ®                Infusion                                                                      10 min 106.5 ± 15.1** 48.1 ± 2.2 7.37 ± 0.02 28.4 ± 1.7                                                           94.1 ± 3.6*   77                                                           ± 10* 50 ± 6 75                                                         ± 4 69 ± 6                                                               at end of 120.3 ±                                                         19**   46.3 ± 2.2                                                          7.40 ± 0.02 29.1                                                           ± 1.9 95.0 ± 3.2                                                         91 ± 13* 54 ± 6                                                        69 ± 4 64 ± 4                                                            infusion                     After Infusion                                                                30 min 126.1 ± 25.0** 47.3 ± 1.8 7.43 ± 0.01 30.9 ± 1.3                                                           93.0 ± 4.7   88 ±                                                       13* 55 ± 8 68 ± 3                                                       63 ± 4                     60 min 116.0 ± 22.2*  48.1 ± 3.4 7.43 ± 0.02 31.2 ± 2.1                                                           92.6 ± 2.9   85 ±                                                       11* 48 ± 7 69 ± 4                                                       66 ± 5                     90 min 137.6 ± 26.8** 52.6 ± 5.0 7.41 ± 0.02 32.1 ± 1.8                                                           98.0 ± 0.5** 85 ±                                                       7* 53 ± 8 71 ± 5                                                        74 ± 8                     120 min 138.8 ± 25.0** 56.2 ± 5.4 7.39 ± 0.02 32.8 ± 1.6                                                          97.8 ± 0.6** 86 ±                                                       6* 53 ± 4 74 ± 5                                                        76 ± 7                     150 min 134.9 ± 26.6** 54.7 ± 5.4 30.8 ± 1.5  30.8 ± 1.5                                                          98.2 ± 0.4** 81 ±                                                       6* 49 ± 5 77 ± 6                                                        79 ± 9                   __________________________________________________________________________     All values are means ± SE, n = 8, *p < 0.05 and **p < 0.01, baseline       values during oxygen breathing versus values during and after EchoGen         ® infusion. PaO.sub.2 -- oxygen tension in arterial blood; PaCO.sub.2     -- carbon dioxide tension in arterial blood, pH and [HCO.sub.3 ] measured     in arterial blood; O.sub.2 sat -- oxygen saturation in arterial blood;        PO.sub.2 K and  #PO.sub.2 R -- oxygen tension in abdominal muscle measure     with Kontron's and Radiometer's transcutaneous combisensors, respectively     PCO.sub.2 K and PCO.sub.2 R -- carbon dioxide tension in abdominal muscle     measured with Kontron's and Radiometer's transcutaneous combisensors,         respectively.                                                            

It should be understood that the embodiments and the examples of thepresent invention, as described herein, are for purposes of illustrationonly, and not limitation, and any changes or modifications as willbecome apparent to one of ordinary skill in the art from the foregoingdescription and accompanying figures are intended to be included withinthe scope of the appended claims and the equivalents thereof.

What is claimed is:
 1. A method for reducing the effects of right toleft circulatory shunt comprising the steps of introducing into a bloodvessel of an individual in need of treatment a therapeutically-effectiveamount of stabilized microbubbles.
 2. The method according to claim 1,further comprising administering oxygen during treatment with thestabilized microbubbles.